Sphingosine kinase enzyme

ABSTRACT

The present invention relates generally to novel protein molecules and to derivatives, analogues, chemical equivalents and mimetics thereof capable of modulating cellular activity and, in particular, modulating cellular activity via the modulation of signal transduction. More particularly, the present invention relates to human sphingosine kinase and to derivatives, analogues, chemical equivalents and mimetics thereof. The present invention also contemplates genetic sequences encoding said protein molecules and derivatives, analogues, chemical equivalents and mimetics thereof. The molecules of the present invention are useful in a range of therapeutic, prophylactic and diagnostic application.

REFERENCE TO RELATED APPLICATIONS

The present application is the national stage under 35 U.S.C. 371 ofinternational application PCT/AU00/00457, filed May 12, 2000 whichdesignated the United States, and which international application waspublished under PCT Article 21(2) in the English language.

FIELD OF THE INVENTION

The present invention relates generally to novel protein molecules andto derivatives, analogues, chemical equivalents and mimetics thereofcapable of modulating cellular activity and, in particular, modulatingcellular activity via the modulation of signal transduction. Moreparticularly, the present invention relates to human sphingosine kinaseand to derivatives, analogues, chemical equivalents and mimeticsthereof. The present invention also contemplates genetic sequencesencoding said protein molecules and derivatives, analogues, chemicalequivalents and mimetics thereof. The molecules of the present inventionare useful in a range of therapeutic, prophylactic and diagnosticapplications.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

Sphingosine kinase is a key regulatory enzyme in a variety of cellularresponses. Its activity can affect inflammation, apoptosis and cellproliferation, and thus it is an important target for therapeuticintervention.

Sphingosine-1-phosphate is known to be an important second messenger insignal transduction (Meyer et al., 1997). It is mitogenic in variouscell types (Alessenko, 1998; Spiegel et al., 1998) and appears totrigger a diverse range of important regulatory pathways including;prevention of ceramide-induced apoptosis (Culliver et al., 1996),mobilisation of intracellular calcium by an IP₃-independant pathway,stimulation of DNA synthesis, activation of mitogen-activated protein(MAP) kinase pathway, activation of phospholipase D, and regulation ofcell motility (for reviews see Meyer et al., 1997; Spiegel et al., 1998;Igarashi., 1997). Recent studies (Xia et al., 1998) have shown thatsphingosine-1-phosphate is an obligatory signaling intermediate in theinflammation response of vascular endothelial cells to tumor necrosisfactors-α(TNFα). In spite of its obvious importance, very little isknown of the mechanisms that control cellular sphingosine-1-phosphatelevels. It is known that sphingosine-1-phosphate levels in the cell aremediated largely by its formation from sphingosine by sphingosinekinase, and to a lesser extent by its degradation by endoplasmicreticulum-associated sphingosine-1-phosphate lyase andsphingosine-1-phosphate phosphatase (Spiegel et al., 1998). Basal levelsof sphingosine-1-phosphate in the cell are generally low, but canincrease rapidly and transiently when cells are exposed to mitogenicagents. This response appears correlated with an increase in sphingosinekinase activity in the cytosol and can be prevented by addition of thesphingosine kinase inhibitory molecules N,N-dimethylsphingosine andDL-threo-dihydrosphingosine. This indicates that sphingosine kinase isan important molecule responsible for regulating cellularsphingosine-1-phosphate levels. This places sphingosine kinase in acentral and obligatory role in mediating the effects attributed tosphingosine-1-phosphate in the cell.

Accordingly, there is a need to identify and clone novel sphingosinekinase molecules to facilitate the progression towards the moresensitive control of intracellular signal transduction via, for example,the elucidation of the mechanism controlling the expression andenzymatic activity of sphingosine kinase thereby providing a platformfor the development of interventional therapies to regulate theexpression or activity of sphingosine kinase. In work leading up to thepresent invention the inventors have purified and cloned a novelsphingosine kinase molecule.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

One aspect of the present invention provides an isolated nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence encoding or complementary to a sequence encoding a novelsphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

Another aspect of the present invention provides an isolated nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence encoding or complementary to a sequence encoding a humansphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

Yet another aspect of the present invention provides a nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence encoding, or a nucleotide sequence complementary to anucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:2 or a derivative or mimetic thereof or having atleast about 45% or greater similarity to at least 10 contiguous aminoacids in SEQ ID NO:2.

Still another aspect of the present invention contemplates a nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence substantially as set forth in SEQ ID NO:1 or a derivativethereof or capable of hybridising to SEQ ID NO:1 under low stringencyconditions.

Still yet another aspect of the present invention contemplates a nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence substantially as set forth in SEQ ID NO:1 or a derivativethereof, or capable of hybridising to SEQ ID NO:1 under low stringencyconditions and which encodes an amino acid sequence corresponding to anamino acid sequence set forth in SEQ ID NO:2 or a sequence having atleast about 45% similarity to at least 10 contiguous amino acids in SEQID NO:2.

A further aspect of the present invention contemplates a nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:1.

Another further aspect of the present invention contemplates a genomicnucleic acid molecule or derivative or analogue thereof capable ofhybridising to SEQ ID NO:1 or a derivative thereof under low stringencyconditions at 42° C.

Still another further aspect of the present invention provides a cDNAsequence comprising a sequence of nucleotides as set forth in SEQ IDNO:1 or a derivative or analogue thereof including a nucleotide sequencehaving similarity to SEQ ID NO:1.

Yet another further aspect of the present invention provides an aminoacid sequence set forth in SEQ ID NO:2 or a derivative, analogue orchemical equivalent or mimetic thereof as defined above or a derivativeor mimetic having an amino acid sequence of at least about 45%similarity to at least 10 contiguous amino acids in the amino acidsequence as set forth in SEQ ID NO:2 or a derivative or mimetic thereof.

Still yet another further aspect of the present invention is directed toan isolated protein selected from the list consisting of:

(i) A novel sphingosine kinase protein or a derivative, analogue,chemical equivalent or mimetic thereof.

(iii) A protein having an amino acid sequence substantially as set forthin SEQ ID NO:2 or a derivative or mimetic thereof or a sequence havingat least about 45% similarity to at least 10 contiguous amino acids inSEQ ID NO:2 or a derivative, analogue, chemical equivalent or mimetic ofsaid protein.

(iv) A protein encoded by a nucleotide sequence substantially as setforth in SEQ ID NO:1 or a derivative or analogue thereof or a sequenceencoding an amino acid sequence having at least about 45% similarity toat least 10 contiguous amino acids in SEQ ID NO:2 or a derivative,analogue, chemical equivalent or mimetic of said protein.

(v) A protein encoded by a nucleic acid molecule capable of hybridisingto the nucleotide sequence as set forth in SEQ ID NO:1 or a derivativeor analogue thereof under low stringency conditions and which encodes anamino acid sequence substantially as set forth in SEQ ID NO:2 or aderivative or mimetic thereof or an amino acid sequence having at leastabout 45% similarity to at least 10 contiguous amino acids in SEQ IDNO:2

(ii) A human sphingosine kinase protein or a derivative, analogue,chemical equivalent or mimetic thereof.

(vi) A protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or(v) in a hormodimeric form.

(vii) A protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or(v) in a heterodimeric form.

Another aspect of the present invention contemplates a method ofmodulating activity of sphingosine kinase in a mammal, said methodcomprising administering to said mammal a modulating effective amount ofan agent for a time and under conditions sufficient to increase ordecrease sphingosine kinase activity.

Still another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to modulate the expression ofa nucleotide sequence encoding sphingosine kinase or sufficient tomodulate the activity of sphingosine kinase.

Yet another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount ofsphingosine kinase or sphingosine kinase.

Still yet another aspect of the present invention relates to a method oftreating a mammal said method comprising administering to said mammal aneffective amount of an agent for a time and under conditions sufficientto modulate the expression of sphingosine kinase or sufficient tomodulate the activity of sphingosine kinase wherein said modulationresults in modulation of cellular functional activity.

A further aspect of the present invention relates to a method oftreating a mammal said method comprising administering to said mammal aneffective amount of sphingosine kinase or sphingosine kinase for a timeand under conditions sufficient to modulate cellular functionalactivity.

Yet another further aspect of the present invention relates to the useof an agent capable of modulating the expression of sphingosine kinaseor modulating the activity of sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

A further aspect of the present invention relates to the use ofsphingosine kinase or sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

Still yet another aspect of the present invention relates to agents foruse in modulating sphingosine kinase expression or sphingosine kinaseactivity wherein said modulation results in modulation of cellularfunctional activity.

Another aspect of the present invention relates to sphingosine kinase orsphingosine kinase for use in modulating cellular functional activity.

In a related aspect of the present invention, the mammal undergoingtreatment may be a human or an animal in need of therapeutic orprophylactic treatment.

In yet another further aspect the present invention contemplates apharmaceutical composition comprising sphingosine kinase, sphingosinekinase or an agent capable of modulating sphingosine kinase expressionor sphingosine kinase activity together with one or morepharmaceutically acceptable carriers and/or diluents. Sphingosinekinase, sphingosine kinase or said agent are referred to as the activeingredients.

Yet another aspect of the present invention contemplates a method fordetecting sphingosine kinase or sphingosine kinase mRNA in a biologicalsample from a subject said method comprising contacting said biologicalsample with an antibody specific for sphingosine kinase or sphingosinekinase mRNA or its derivatives or homologous for a time and underconditions sufficient for an antibody-sphingosine kinase orantibody-sphingosine kinase mRNA complex to form, and then detectingsaid complex.

Single and three letter abbreviations used throughout the specificationare defined in Table 1.

TABLE 1 Single and three letter amino acid abbreviations Three-letterOne-letter Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln QGlutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine The T Tryptophan Trp W Tyrosine Tyr YValine Val V Any residue Xaa X

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the ‘Sphingomyelin pathway’.

FIG. 2 is a graphical representation of anion exchange chromatography ofhuman sphingosine kinase. A, Anion exchange chromatography with QSepharose fast flow of the (NH₄)₂SO₄ precipitated fraction of the humanplacenta extract showing two peaks of sphingosine kinase activity.Extracts were applied in buffer A and sphingosine kinase activity ()was eluted with a NaCl gradient of 0 to 1M (----). Protein eluted wasfollowed by absorbance at 280 nm (—).

FIG. 3 is a graphical representation of the purification of humanplacenta SK1 by anion exchange chromatography. Purification on a Mono-Qcolumn is enhanced by reapplication and elution from the same column inthe presence of ATP. A, The fractions from the calmodulin-Sepharose 4bcolumn containing sphingosine kinase activity were pooled, desalted andapplied to the Mono-Q column in buffer A. B, Active fractions from theMono-Q column were desalted and reapplied to the Mono-Q column in bufferA with: 1 mM ATP and 4 mM MgCl₂. In both cases sphingosine kinaseactivity () was eluted with a NaCl gradient of 0 to 1M(----), withprotein elution followed by absorbance at 280 nm (—).

FIG. 4 is an image of SDS-PAGE of purified human placenta sphingosinekinase. The fraction from the Superdex 75 column containing the highestsphingosine kinase activity was applied to SDS-PAGE with silverstaining, yielding a single band of 45 kDa.

FIG. 5 is a graphical representation of preparative anion exchangechromatography. Only a single chromatographically identified sphingosinekinase isoform is present in HUVEC. Preparative anion exchangechromatography with HiTrap-Q columns of human placenta, HUVEC and TNFαtreated HUVEC extracts showing, in HUVEC, the presence of a singlesphingosine kinase peak that increases in activity following treatmentof cells with TNFα. Cells were harvested, lysed and the soluble extractsapplied to the HiTrap-Q column in buffer A. Total sphingosine kinaseactivity in human placenta, HUVEC and TNFα treated HUVEC extracts were51, 78 and 136 U/mg proteins respectively Sphingosine kinase activity(□) was eluted with a NaCl gradient of 0 to 1M (----).

FIG. 6 is a schematic representation of the strategy used to clonesphingosine kinase (SPHK) from HUVEC.

FIG. 7 is a schematic representation of the nucleotide (nucleotides15-1187 of SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequencesand putative domain structure of human sphingosine kinase, a(i)-a(ii),cDNA nucleic acid sequence and the deduced amino acid sequence of hSK.Amino acids are numbered from the first methionine residue. The stopcodon is indicated by an asterisk. The sphingosine kinase coding regionis in capital letters (nucleotides 33-1187), while lower case lettersdenote untranslated and vector sequence. b, Schematic representation ofhuman sphingosine kinase showing locations of the putative PKC and CKIIphosphorylation sites, a possible N-myristoylation site,calcium/calmodulin binding motifs and the region with similarity to theputative DGK catalytic domain.

FIG. 8 is a graphical representation of the expression and TNFαstimulation of human sphingosine kinase activity in HEK293 cells. HEK293cells transiently transfected with either empty pcDNA expression vectoralone (A) or pcDNA containing human sphingosine kinase cDNA (B).Transfected cells were either untreated or treated with TNFα for 10 min,harvested and sphingosine kinase activity in cell lysates determined.Data are means of duplicates and are representative of three independentexperiments.

FIG. 9 (i)-(iv) is a schematic representation of the sequence comparisonof human sphingosine kinase with other known and putative sphingosinekinases. Comparison of the deduced human sphingosine kinase amino acidsequence with the amino acid sequences of the murine (mSK1a (SEQ IDNOs:9-16) and mSK1b (SEQ ID NOs:17-24); Kohama et al., 1998) and S.cerevisiae (LCB4 (SEQ ID NOs:25-32) and LCB5 (SEQ ID NOs:33-40); Nagiecet al., 1998) sphingosine kinases, and EST sequences of putativesphingosine kinases from S. pombe (SEQ ID NOs:41-48) and C. elegans (SEQID NOs:49-56) (Genbank™ accession numbers Z98762 and Z66494,respectively). Although the amino acid sequence similarity to humansphingosine kinase was high (36% identity), the A. thaliana putativesphingosine kinase sequence (Genbank™ accession number AL022603) gaverelatively poor alignment and, for clarity, is not shown. The consensussequence represents amino acids that are conserved in at least six ofthe seven aligned sequences, while conservation of structurally similaramino acids are denoted with an asterisk. Multiple sequence alignmentwas performed with CLUSTALW, and percentage identities to the humansphingosine kinase were determined using the GAP algorithm (Needleman &Wunsch, 1970). number AL022603) gave relativey poor alignment and, forclarity, is not shown. The consensus sequence represents amino acidsthat are conserved in at least six of the seven aligned sequences, whileconservation of structurally similar amino acids are denoted with anasterisk. Multiple sequence alignment was performed with CLUSTALW, andpercentage identities to the human sphingosine kinase were determinedusing the GAP algorithm (Needleman & Wunsch, 1970).

FIG. 10 is an image of the expression of recombinant human sphingosinekinase in E. coli BL21. E. coli BL21 was transformed with the pGEX4-2Thuman sphingosine kinase expression construct and expression of theGST-SK fusion protein analysed after induction with 100 μM IPTG. A,Sphingosine kinase activity in uninduced and IPTG induced E. coli BL21cell extracts. B, Coomassie stained SDS-PAGE gel showing GST-SK fusionprotein expression in E. coli cell lysates. C, Purity of the isolatedrecombinant human sphingosine kinase. The fraction from the Mono-Qcolumn containing sphingosine kinase activity was applied to SDS-PAGEwith silver staining yielding a single band of 45 kDa.

FIG. 11 is an image of the purification of recombinant human sphingosinekinase. Calmodulin Sepharose 4B allows the separation of active andinactive enzyme. The GST-SK fusion protein was partially purified usingglutathione-Sepharose 4B, cleaved by thrombin and applied to thecalmodulin-Sepharose 4B column in Buffer B containing 4 mM CaCl₂.Elution (1) of the active sphingosine kinase bound to the column wasperformed with Buffer A containing 2 mM EGTA and 1 M NaCl. A, SDS-PAGEanalysis of fractions eluted from the calmodulin-Sepharose 4B column. B,Sphingosine kinase activity (|) in column fractions showing most of therecombinant human sphingosine kinase protein did not bind to the columnand displayed no catalytic activity.

FIG. 12 is a graphical representation of the physico-chemical propertiesof the native and recombinant sphingosine kinases. A, pH optima. Theeffect of pH on SK activity was determined by assaying the activity overthe pH range of 4 to 11 in 50 mM buffers (sodium acetate, pH 4.0-5.0;Mes, pH 6.0-7.0; Hepes, pH 7.0-8.2; Tris/HCl, pH 8.2-10.0; Caps,10.0-11.0). B, pH stability. Data shown is the SK activity remainingafter preincubation of the enzymes at various pH at 4° C. for 5h. C.Temperature stability. Data shown is the SK activity remaining afterpreincubation of the enzymes at various temperatures (4 to 80° C.) for30 min at pH 7.4 (50 mM Tris/HCl containing 10% glycerol. 0.5 M NaCl and0.05% Triton X-100). D, Metal ion requirement. The various metal ions orEDTA were supplied in the assay mixture at a final concentration of 10mM. In all cases the maximum activities of the native (□ and filledbars) and recombinant (□ and open bars) sphingosine kinases werearbitrarily set at 100% and correspond to 2.65 kU and 7.43 kU,respectively. Data are means ±S.D.

FIG. 13 is a graphical representation of the substrate specificity andkinetics of the native and recombinant sphingosine kinases. A. Substratespecificity of the native (filled bars) and recombinant (open bars)sphingosine kinases with sphingosine analogues and other lipids suppliedat 100 μM in 0.25% Triton X-100. The rates of phosphorylation ofsphingosine by the native and recombinant Sks were arbitrarily set at100% and correspond to 2.65 kU and 7.43 kU, respectively. Activityagainst other potential substrates were expressed relative to theactivity against sphingosine. No phosphorylation was observed withDL-threo-dihydrosphingosine, N,N-dimethylsphingosine, N,N,N,-trimethylsphingosinei N-acetylsphingosine (C₂-ceramide), diacylglycerol(1,2-dioctanoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol), andphosphatidylinositol. B, Substrate kinetics of the recombinant humansphingosine kinase with sphingosine (□) and D-erythro-dihydrosphingosine(□) as substrates. C, Kinetics if inhibition of the recombinant humansphingosine kinase with N,N,N-trimethylsphingosine at 5 μM (□) and 25 μM(□), and in the absence of N,N,N-trimethylsphingosine (□). Inset:Lineweaver-Burk plot. Data are means ±S.D.

FIG. 14 is a graphical representation of the acidic phospholipids whichstimulate the activity of the native and recombinant sphingosinekinases. The effect of various phospholipids on the activity of thenative and recombinant sphingosine kinases were determined by assayingthe activity under standard conditions in the presence if thesephospholipids at 10 mol% of Triton X-100. The activities of the native(filled bars) and recombinant (open bars) sphingosine kinases in theabsence of phospholipids were arbitrarily set at 100% and correspond to2.65 kU and 7.43 kU, respectively. PC, phosphatidylcholine; PS,phosphatidyiserine; PE, phosphatidylethanolamine: PIphosphatidylinositol; PA, phosphatidic acid. Data are means=S.D.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the purification andcloning of a novel sphingosine kinase molecule. The identification ofthis novel molecule permits the identification and rational design of arange of products for use in therapy, diagnosis and antibody generation,for example for use in signal transduction. These therapeutic moleculesmay also act as either antagonists or agonists of sphingosine kinasefunction and will be useful, infer alia, in the modulation of cellularactivation in the treatment of disease conditions characterised byunwanted cellular activity.

Accordingly, one aspect of the present invention provides an isolatednucleic acid molecule or derivative or analogue thereof comprising anucleotide sequence encoding or complementary to a sequence encoding anovel sphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

Reference to “sphingosine kinase” should be understood as a reference tothe molecule which is, inter alia, involved in the generation ofsphingosine-1-phosphate during the activation of the sphingosine kinasesignalling pathway. Reference to “sphingosine kinase” in italicised textshould be understood as a reference to the sphingosine kinase nucleicacid molecule. Reference to “sphingosine kinase” in non-italicised textshould be understood as a reference to the sphingosine kinase proteinmolecule.

More particularly, the present invention provides an isolated nucleicacid molecule or derivative or analogue thereof comprising a nucleotidesequence encoding of complementary to a sequence encoding a humansphingosine kinase protein or a derivative or mimetic of saidsphingosine kinase protein.

In a preferred embodiment, the present invention provides a nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence encoding, or a nucleotide sequence complementary to anucleotide sequence encoding, an amino acid sequence substantially asset forth in SEQ ID NO:2 or a derivative or mimetic thereof or having atleast about 45% or greater similarity to at least 10 contiguous aminoacids in SEQ ID NO:2. substantially as set forth in <400>2 or aderivative or mimetic thereof or having at least about 45% or greatersimilarity to at least 10 contiguous amino acids in <400>2.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. The percentage similarity maybe greater than 50% such as at least 70% or at least 80% or at least 90%or at least 95% or higher.

Another aspect of the present invention contemplates a nucleic acidmolecule or derivative or analogue thereof comprising a nucleotidesequence substantially as set forth in SEQ ID NO:1 or a derivativethereof, or capable of hybridising to SEQ ID NO:1 under low stringencyconditions.

Reference herein to a low stringency includes and encompasses from atleast about 0% v/v to at least about 15% v/v formamide and from at leastabout 1M to at least about 2M salt for hybridisation, and at least about1M to at least about 2M salt for washing conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5M to atleast about 0.9M salt for hybridisation, and at least about 0.5M to atleast about 0.9M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01M to at least about 0.15Msalt for hybridisation, and at least about 0.01M to at least about 0.15Msalt for washing conditions. Stringency may be measured using a range oftemperature such as from about 40° C. to about 65° C. Particularlyuseful stringency conditions are at 42° C. In general, washing iscarried out at T_(m)=69.3+0.41 (G+C)%[19]=−12° C. However, the T_(m) ofa duplex DNA decreases by 1° C. with every increase of 1% in the numberof mismatched based pairs (Bonner et al (1973) J.Mol.Biol, 81:123).

Preferably, the present invention contemplates a nucleic acid moleculeor derivative or analogue thereof comprising a nucleotide sequencesubstantially as set forth in SEQ ID NO:1 or a derivative thereof orcapable of hybridising to SEQ ID NO:1 under low stringency conditionsand which encodes an amino acid sequence corresponding to an amino acidsequence set forth in SEQ ID NO:2 or a sequence having at least about45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2.

More particularly, the present invention contemplates a nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:1.

The nucleic acid molecule according to this aspect of the presentinvention corresponds herein to human sphingosine kinase. Withoutlimiting the present invention to any one theory or mode of action, theprotein encoded by sphingosine kinase is a key element in thefunctioning of the sphingosine kinase signalling pathway. Sphingosinekinase acts to facilitate the generation of the second messenger,sphingosine-1-phosphate, and may be activated by:

(a) post-translational modifications such as phosphorylation orproteolytic cleavage;

(b) protein-protein interactions such as dimerisation, and Gprotein-coupled receptor mediated interactions;

(c) translocational events where the enzyme is targeted to anenvironment that increases catalytic activity or allows access to itssubstrate.

The expression product of the human sphingosine kinase nucleic acidmolecule is human sphingosine kinase. Sphingosine kinase is defined bythe amino acid sequence set forth in SEQ ID NO:2. The cDNA sequence forsphingosine kinase is defined by the nucleotide sequence set forth inSEQ ID NO:1. The nucleic acid molecule encoding sphingosine kinase ispreferably a sequence of deoxyribonucleic acids such as a cDNA sequenceor a genomic sequence. A genomic sequence may also comprise exons andintrons. A genomic sequence may also include a promoter region or otherregulatory regions.

Another aspect of the present invention contemplates a genomic nucleicacid molecule or derivative thereof capable of hybridising to SEQ IDNO:1 or a derivative thereof under low stringency conditions at 42° C.

Reference herein to sphingosine kinase and sphingosine kinase should beunderstood as a reference to all forms of human sphingosine kinase andsphingosine kinase, respectfully, including, for example, any peptideand cDNA isoformns which arise from alternative splicing of sphingosinekinase mRNA, mutants or polymorphic variants of sphingosine kinase orsphingosine kinase, the post-translation modified form of sphingosinekinase or the non-post-translation modified form of sphingosine kinase.To the extent that it is not specified, reference herein to sphingosinekinase and sphingosine kinase includes reference to derivatives,analogues, chemical equivalents and mimetics thereof.

The protein and/or gene is preferably from the human. However, theprotein and/or gene may also be isolated from other animal or non-animalspecies.

Derivatives include fragments, parts, portions, mutants, variants andmimetics from natural, synthetic or recombinant sources including fusionproteins. Parts or fragments include, for example, active regions ofsphingosine kinase. Derivatives may be derived from insertion, deletionor substitution of amino acids. Amino acid insertional derivativesinclude amino and/or carboxylic terminal fusions as well asintrasequence insertions of single or multiple amino acids. Insertionalamino acid sequence variants are those in which one or more amino acidresidues are introduced into a predetermined site in the proteinalthough random insertion is also possible with suitable screening ofthe resulting product. Deletional variants are characterized by theremoval of one or more amino acids from the sequence. Substitutionalamino acid variants are those in which at least one residue in thesequence has been removed and a different residue inserted in its place.An example of substitutional amino acid variants are conservative aminoacid substitutions. Conservative amino acid substitutions typicallyinclude substitutions within the following Groups: glycine and alanine;valine, isoleucine and leucine; aspartic acid and glutamic acid;asparagine and glutamine; serine and threonine; lysine and arginine; andphenylalanine and tyrosine. Additions to amino acid sequences includingfusions with other peptides, polypeptides or proteins.

Chemical and functional equivalents of sphingosine kinase or sphingosinekinase should be understood as molecules exhibiting any one or more ofthe functional activities of sphingosine kinase or sphingosine kinaseand may be derived from any source such as being chemically synthesizedor identified via screening processes such as natural product screening.

The derivatives of sphingosine kinase include fragments havingparticular epitopes or parts of the entire sphingosine kinase proteinfused to peptides, polypeptides or other proteinaceous ornon-proteinaceous molecules.

Analogues of sphingosine kinase contemplated herein include, but are notlimited to, modification to side chains, incorporating of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the proteinaceous molecules ortheir analogues.

Derivatives of nucleic acid sequences may similarly be derived fromsingle or multiple nucleotide substitutions, deletions and/or additionsincluding fusion with other nucleic acid molecules. The derivatives ofthe nucleic acid molecules of the present invention includeoligonucleotides, PCR primers, antisense molecules, molecules suitablefor use in cosuppression and fusion of nucleic acid molecules.Derivatives of nucleic acid sequences also include degenerate variants.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzenesuiphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds reaction with maleimide, maleic anhydride or other substitutedmaleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides; Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acid contemplated herein is show in Table 2.

TABLE 2 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtnp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-a-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety.

The nucleic acid molecule of the present invention is preferably inisolated form or ligated to a vector, such as an expression vector. By“isolated” is meant a nucleic acid molecule having undergone at leastone purification step and this is conveniently defined, for example, bya composition comprising at least about 10% subject nucleic acidmolecule, preferably at least about 20%, more preferably at least about30%, still more preferably at least about 40-50%, even still morepreferably at least about 60-70%, yet even still more preferably 80-90%or greater of subject nucleic acid molecule relative to other componentsas determined by molecular weight, encoding activity, nucleotidesequence, base composition or other convenient means. The nucleic acidmolecule of the present invention may also be considered, in a preferredembodiment, to be biologically pure.

The term “protein” should be understood to encompass peptides,polypeptides and proteins. The protein may be glycosylated orunglycosylated and/or may contain a range of other molecules fused,linked, bound or otherwise associated to the protein such as aminoacids, lipids, carbohydrates or other peptides, polypeptides orproteins. Reference hereinafter to a “protein” includes a proteincomprising a sequence of amino acids as well as a protein associatedwith other molecules such as amino acids, lipids, carbohydrates or otherpeptides, polypeptides or proteins.

In a particularly preferred embodiment, the nucleotide sequencecorresponding to sphingosine kinase is a cDNA sequence comprising asequence of nucleotides as set forth in SEQ ID NO:1 or a derivative oranalogue thereof including a nucleotide sequence having similarity toSEQ ID NO:1.

A derivative of a nucleic acid molecule of the present invention alsoincludes a nucleic acid molecule capable of hybridising to a nucleotidesequence as set forth in SEQ ID NO:1 under low stringency conditions.Preferably, low stringency is at 42° C.

The nucleic acid molecule may be ligated to an expression vector capableof expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell(e.g. yeast cells, fungal cells, insect cells, mammalian cells or plantcells). The nucleic acid molecule may be ligated or fused or otherwiseassociated with a nucleic acid molecule encoding another entity such as,for example, a signal peptide. It may also comprise additionalnucleotide sequence informnation fused, linked or otherwise associatedwith it either at the 3′ or 5′ terminal portions or at both the 3′ and5′ terminal portions. The nucleic acid molecule may also be part of avector, such as an expression vector. The latter embodiment facilitatesproduction of recombinant forms of sphingosine kinase which forms areencompassed by the present invention.

The present invention extends to the expression product of the nucleicacid molecules as hereinbefore defined.

The expression product is sphingosine kinase having an amino acidsequence set forth in SEQ ID NO:2 or is a derivative, analogue orchemical equivalent or mimetic thereof as defined above or is aderivative or mimetic having an amino acid sequence of at least about45% similarity to at least 10 contiguous amino acids in the amino acidsequence as set forth in <400>2 or a derivative or mimetic thereof.

Another aspect of the present invention is directed to an isolatedprotein selected from the list consisting of:

(i) A novel sphingosine kinase protein or a derivative, analogue,chemical equivalent or mimetic thereof.

(ii) A human sphingosine kinase protein or a derivative, analogue,chemical equivalent or mimetic thereof

(iii) A protein having an amino acid sequence substantially as set forthin SEQ ID NO:2 or a derivative or mimetic thereof or a sequence havingat least about 45% similarity to at least 10 contiguous amino acids inSEQ ID NO:2 or a derivative, analogue, chemical equivalent or mimetic ofsaid protein.

(iv) A protein encoded by a nucleotide sequence substantially as setforth in SEQ ID NO:1 or a derivative or analogue thereof or a sequenceencoding an amino acid sequence having at least about 45% similarity toat least 10 contiguous amino acids in SEQ ID NO:2 or a derivative,analogue, chemical equivalent or mimetic of said protein.

(v) A protein encoded by a nucleic acid molecule capable of hybridisingto the nucleotide sequence as set forth in SEQ ID NO:1 or a derivativeor analogue thereof under low stringency conditions and which encodes anamino acid sequence substantially as set forth in SEQ ID NO:2 or aderivative or mimetic thereof or an amino acid sequence having at leastabout 45% similarity to at least 10 contiguous amino acids in SEQ IDNO:2.

vi) A protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or(v) in a homodimeric form.

vii) A protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or(v) in a heterodimeric form.

The protein of the present invention is preferably in isolated form. By“isolated” is meant a protein having undergone at least one purificationstep and this is conveniently defined, for example, by a compositioncomprising at least about 10% subject protein, preferably at least about20%, more preferably at least about 30%, still more preferably at leastabout 40-50%, even still more preferably at least about 60-70%, yet evenstill more preferably 80-90% or greater of subject protein relative toother components as determined by molecular weight, amino acid sequenceor other convenient means. The protein of the present invention may alsobe considered, in a preferred embodiment, to be biologically pure.

The sphingosine kinase of the present invention may be in multimericform meaning that two or more molecules are associated together. Wherethe same sphingosine kinase molecules are associated together thecomplex is a homomultimer. An example of a homomultimer is a homodimer.Where at least one sphingosine kinase is associated with at least onenon-sphingosine kinase molecule, then the complex is a heteromultimersuch as a heterodimer.

The ability to produce recombinant sphingosine kinase permits the largescale production of sphingosine kinase for commercial use. Thesphingosine kinase may need to be produced as part of a large peptide,polypeptide or protein which may be used as is or may first need to beprocessed in order to remove the extraneous proteinaceous sequences.Such processing includes digestion with proteases, peptidases andamidases or a range of chemical, electrochemical, sonic or mechanicaldisruption techniques.

Notwithstanding that the present invention encompasses recombinantproteins, chemical synthetic techniques are also preferred in synthesisof sphingosine kinase.

Sphingosine kinase according to the present invention is convenientlysynthesised based on molecules isolated from the human. Isolation of thehuman molecules may be accomplished by any suitable means such as bychromotographic separation, for example using CM-cellulose ion exchangechromatography followed by Sephadex (e.g. G-50 column) filtration. Manyother techniques are available including HPLC, PAGE amongst others.

Sphingosine kinase may be synthesised by solid phase synthesis usingF-moc chemistry as described by Carpino et al. (1991). Sphingosinekinase and fragments thereof may also be synthesised by alternativechemistries including, but not limited to, t-Boc chemistry as describedin Stewart et al. (1985) or by classical methods of liquid phase peptidesynthesis.

Without limiting the theory or mode of action of the present invention,sphingosine kinase is a key regulatory enzyme in the activity of thesphingosine kinase signalling pathway. By “sphingosine kinase signallingpathway” is meant a signalling pathway which utilises one or both ofsphingosine kinase and/or sphingosine-1-phosphate. It is thought that asphingosine kinase signalling pathway cascade which results in adhesionmolecule expression may take the form of:

(i) the generation of ceramide from sphingomyelin via S.Mase activity,said ceramide being converted to sphingosine;

(ii) sphingosine-1-phosphate (referred to hereinafter as “Sph-1-P”)generation by stimulation of sphingosine kinase; and

(iii) the activation of MEK/ERK and nuclear translocation of NF-κBdownstream from Sph-1-P generation.

The sphingosine kinase signalling pathway is known to regulate cellularactivities such as those which lead to inflammation, apoptosis and cellproliferation. For example, upregulation of the production ofinflammatory mediators such as cytokines, chemokines, eNOS andupregulation of adhesion molecule expression. Said upregulation may beinduced by a number of stimuli including, for example, inflammatorycytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1(IL-1), endotoxin, oxidised or modified lipids, radiation or tissueinjury.

The cloning and sequencing of this gene and its expression product nowprovides additional molecules for use in the prophylactic andtherapeutic treatment of diseases characterised by unwanted cellularactivity, which activity is either directly or indirectly modulated viathe activity of the sphingosine kinase signalling pathway. Examples ofdiseases involving unwanted sphingosine kinase regulated cellularactivity include rheumatoid arthritis, asthma, atherosclerosis,meningitis, multiple sclerosis and septic shock. Accordingly, thepresent invention contemplates therapeutic and prophylactic uses ofsphingosine kinase amino acid and nucleic acid molecules, in addition tosphingosine kinase agonistic and antagonistic agents, for the regulationof cellular functional activity, such as for example, regulation ofinflammation.

The present invention contemplates, therefore, a method for modulatingexpression of sphingosine kinase in a subject, said method comprisingcontacting the sphingosine kinase gene with an effective amount of anagent for a time and under conditions sufficient to up-regulate ordown-regulate or otherwise modulate expression of sphingosine kinase.For example, sphingosine kinase antisense sequences such asoligonucleotides may be introduced into a cell to down-regulate one ormore specific functional activities of that cell. Conversely, a nucleicacid molecule encoding sphingosine kinase or a derivative thereof may beintroduced to up-regulate one or more specific functional activities ofany cell not expressing the endogenous sphingosine kinase gene.

Another aspect of the present invention contemplates a method ofmodulating activity of sphingosine kinase in a mammal, said methodcomprising administering to said mammal a modulating effective amount ofan agent for a time and under conditions sufficient to increase ordecrease sphingosine kinase activity.

Modulation of said activity by the administration of an agent to amammal can be achieved by one of several techniques, including but in noway limited to introducing into said mammal a proteinaceous ornon-proteinaceous molecule which:

(i) modulates expression of sphingosine kinase;

(ii) functions as an antagonist of sphingosine kinase;

(iii) functions as an agonist of sphingosine kinase.

Said proteinaceous molecule may be derived from natural or recombinantsources including fusion proteins or following, for example, naturalproduct screening. Said non-proteinaceous molecule may be, for example,a nucleic acid molecule or may be derived from natural sources, such asfor example natural product screening or may be chemically synthesised.The present invention contemplates chemical analogs of sphingosinekinase or small molecules capable of acting as agonists or antagonistsof sphingosine kinase. Chemical agonists may not necessarily be derivedfrom sphingosine kinase but may share certain conformationalsimilarities. Alternatively, chemical agonists may be specificallydesigned to mimic certain physiochemical properties of sphingosinekinase. Antagonists may be any compound capable of blocking, inhibitingor otherwise preventing sphingosine kinase from carrying out its normalbiological functions. Antagonists include monoclonal antibodies specificfor sphingosine kinase, or parts of sphingosine kinase, and antisensenucleic acids which prevent transcription or translation of sphingosinekinase genes or mRNA in mammalian cells. Modulation of sphingosinekinase expression may also be achieved utilizing antigens, RNA,ribosomes, DNAzymes, RNA aptamers or antibodies.

Said proteinaceous or non-proteinaceous molecule may act either directlyor indirectly to modulate the expression of sphingosine kinase or theactivity of sphingosine kinase. Said molecule acts directly if itassociates with sphingosine kinase or sphingosine kinase to modulate theexpression or activity of sphingosine kinase or sphingosine kinase. Saidmolecule acts indirectly if it associates with a molecule other thansphingosine kinase or sphingosine kinase which other molecule eitherdirectly or indirectly modulates the expression or activity ofsphingosine kinase or sphingosine kinase. Accordingly, the method of thepresent invention encompasses the regulation of sphingosine kinase orsphingosine kinase expression or activity via the induction of a cascadeof regulatory steps which lead to the regulation of sphingosine kinaseor sphingosine kinase expression or activity.

Another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount of an agentfor a time and under conditions sufficient to modulate the expression ofa nucleotide sequence encoding sphingosine kinase or sufficient tomodulate the activity of sphingosine kinase.

Yet another aspect of the present invention contemplates a method ofmodulating cellular functional activity in a mammal said methodcomprising administering to said mammal an effective amount ofsphingosine kinase or sphingosine kincase.

The sphingosine kinase, sphingosine kinase or agent used may also belinked to a targeting means such as a monoclonal antibody, whichprovides specific delivery of the sphingosine kinase, sphingosine kinaseor agent to the target cells.

In a preferred embodiment of the present invention, the sphingosinekinase, sphingosine kinase or agent used in the method is linked to anantibody specific for said target cells to enable specific delivery tothese cells.

Reference to “modulating cellular functional activity” is a reference toup-regulating, down-regulating or otherwise altering any one or more ofthe activities which a cell is capable of performing such as, but notlimited to, one or more of chemokine production, cytokine production,nitric oxide synthesase, adhesion molecule expression and production ofother inflammatory modulators.

Administration of the sphingosine kinase, sphingosine kinase or agent,in the form of a pharmaceutical composition, may be performed by anyconvenient means. Sphingosine kinase, sphingosine kinase or agent of thepharmaceutical composition are contemplated to exhibit therapeuticactivity when administered in an amount which depends on the particularcase. The variation depends, for example, on the human or animal and thesphingosine kinase, sphingosine kinase or agent chosen. A broad range ofdoses may be applicable. Considering a patient, for example, from about0.1 mg to about 1 mg of sphingosine kinase or agent may be administeredper kilogram of body weight per day. Dosage regimes may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily, weekly, monthly or other suitable timeintervals or the dose may be proportionally reduced as indicated by theexigencies of the situation. The sphingosine kinase or agent may beadministered in a convenient manner such as by the oral, intravenous(where water soluble), intrannasal, intraperitoneal, intramuscular,subcutaneous, intradermal or suppository routes or implanting (e.g.using slow release molecules). With particular reference to use ofsphingosine kinase or agent, these peptides may be administered in theform of pharmaceutically acceptable nontoxic salts, such as acidaddition salts or metal complexes, e.g. with zinc, iron or the like(which are considered as salts for purposes of this application).Illustrative of such acid addition salts are hydrochloride,hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate,succinate, malate, ascorbate, tartrate and the like. If the activeingredient is to be administered in tablet form, the tablet may containa binder such as tragacanth, corn starch or gelatin; a disintegratingagent, such as alginic acid; and a lubricant, such as magnesiumstearate.

A further aspect of the present invention relates to the use of theinvention in relation to mammalian disease conditions. For example, thepresent invention is particularly useful, but in no way limited to, usein inflammatory diseases.

Accordingly, another aspect of the present invention relates to a methodof treating a mammal said method comprising administering to said mammalan effective amount of an agent for a time and under conditionssufficient to modulate the expression of sphingosine kinase orsufficient to modulate the activity of sphingosine kinase wherein saidmodulation results in modulation of cellular functional activity.

In another aspect the present invention relates to a method of treatinga mammal said method comprising administering to said mammal aneffective amount of sphingosine kinase or sphingosine kinase for a timeand under conditions sufficient to modulate cellular functionalactivity.

Yet another aspect of the present invention relates to the use of anagent capable of modulating the expression of sphingosine kinase ormodulating the activity of sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

A further aspect of the present invention relates to the use ofsphingosine kinase or sphingosine kinase in the manufacture of amedicament for the modulation of cellular functional activity.

Still yet another aspect of the present invention relates to agents foruse in modulating sphingosine kinase expression or sphingosine kinaseactivity wherein said modulation results in modulation of cellularfunctional activity.

Another aspect of the present invention relates to sphingosine kinase orsphingosine kinase for use in modulating cellular functional activity.

In a related aspect of the present invention, the mammal undergoingtreatment may be a human or an animal in need of therapeutic orprophylactic treatment.

In yet another further aspect the present invention contemplates apharmaceutical composition comprising sphingosine kinase, sphingosinekinase or an agent capable of modulating sphingosine kinase expressionor sphingosine kinase activity together with one or morepharmaceutically acceptable carriers and/or diluents. Sphingosinekinase, sphingosine kinase or said agent are referred to as the activeingredients.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsuperfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal andthe like, In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When sphingosine kinase, sphingosine kinase and sphingosine kinasemodulators are suitably protected they may be orally administered, forexample, with an inert diluent or with an assimilable edible carrier, orthey may be enclosed in hard or soft shell gelatin capsule, or they maybe compressed into tablets, or they may be incorporated directly withthe food of the diet. For oral therapeutic administration, the activecompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such asucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active material and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active material for the treatment ofdisease in living subjects having a diseased condition in which bodilyhealth is impaired.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example contain the principalactive compound in amounts ranging from 0.5 μg to about 2000 mg.Expressed in proportions, the active compound is generally present infrom about 0.5 μg to about 2000 mg/ml of carrier. In the case ofcompositions containing supplementary active ingredients, the dosagesare determined by reference to the usual dose and manner ofadministration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of expressing sphingosinekinase, modulating sphingosine kinase expression or sphingosine kinaseactivity. The vector may, for example, be a viral vector.

Sphingosine kinase can also be utilised to create gene knockout modelsin either cells or animals, which knocked out gene is the sphingosinekinase gene expressed by said cells or animals. Accordingly in anotheraspect the present invention should be understood to extend to methodsof creating sphingosine kinase gene cell or animal knockout modelswherein sphingosine kinase has been utilised to facilitate knocking outof the endogenous sphingosine kinase gene of said cell or animal, and tothe knockout models produced therefrom.

Still another aspect of the present invention is directed to antibodiesto sphingosine kinase including catalytic antibodies. Such antibodiesmay be monoclonal or polyclonal and may be selected from naturallyoccurring antibodies to sphingosine kinase or may be specifically raisedto sphingosine kinase. In the case of the latter, sphingosine kinase mayfirst need to be associated with a carrier molecule. The antibodiesand/or recombinant sphingosine kinase of the present invention areparticularly useful as therapeutic or diagnostic agents.

Altenatively, fragments of antibodies may be used such as Fab fragments.Furthermore, the present invention extends to recombinant and syntheticantibodies and to antibody hybrids. A “synthetic antibody” is consideredherein to include fragments and hybrids of antibodies. The antibodies ofthis aspect of the present invention are particularly useful forimmunotherapy and may also be used as a diagnostic tool, for example,for monitoring the program of a therapeutic regime.

For example, sphingosine kinase can be used to screen for naturallyoccurring antibodies to sphingosine kinase. These may occur, for examplein some inflammatory disorders.

For example, specific antibodies can be used to screen for sphingosinekinase proteins: The latter would be important, for example, as a meansfor screening for levels of sphingosine kinase in a cell extract orother biological fluid or purifying sphingosine kinase made byrecombinant means from culture supernatant fluid. Techniques for theassays contemplated herein are known in the art and include, forexample, sandwich assays, ELISA and flow cytometry.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies) directedto the first mentioned antibodies discussed above. Both the first andsecond antibodies may be used in detection assays or a first antibodymay be used with a commercially available anti-immunoglobulin antibody.An antibody as contemplated herein includes any antibody specific to anyregion of sphingosine kinase.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the protein or peptide derivatives and either type is utilizablefor immunoassays. The methods of obtaining both types of sera are wellknown in the art. Polyclonal sera are less preferred but are relativelyeasily prepared by injection of a suitable laboratory animal with aneffective amount of sphingosine kinase, or antigenic parts thereof,collecting serum from the animal, and isolating specific sera by any ofthe known immunoadsorbent techniques. Although antibodies produced bythis method are utilizable in virtually any type of immunoassays, theyare generally less favored because of the potential heterogeneity of theproduct.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example Douillard and Hoffman, Basic Facts aboutHybridomas, in Compendium of Immunology Vol II, ed. by Schwartz, 1981:Kohler and Milstein, Nature 256: 495-499 1975; European Journal ofImmunology 6: 511-519, 1976).

In another aspect of the present invention, the molecules of the presentinvention are also useful as screening targets for use in applicationssuch as the diagnosis of disorders which are regulated by sphingosinekinase.

Yet another aspect of the present invention contemplates a method fordetecting sphingosine kinase or sphingosine kinase mRNA in a biologicalsample from a subject said method comprising contacting said biologicalsample with an antibody specific for sphingosine kinase or sphingosinekinase mRNA or its derivatives or homologous for a time and underconditions sufficient for an antibody-sphingosine kinase orantibody-sphingosine kinase mRNA complex to form, and then detectingsaid complex.

The presence of sphingosine kinase may be determined in a number of wayssuch as by Western blotting, ELISA or flow cytometry procedures.Sphingosine kinase mRNA may be detected, for example, by in situhybridization or Northern blotting. These, of course, include bothsingle-site and two-site or “sandwich” assays of the non-competitivetypes, as well as in the traditional competitive binding assays. Theseassays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays andare favoured for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabelled antibody is immobilized on a solid substrate andthe sample to be tested brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen complex, a second antibodyspecific to the antigen, labelled with a reporter molecule capable ofproducing a detectable signal is then added and incubated, allowing timesufficient for the formation of another complex ofantibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound antibody. These techniques are well known to those skilled inthe art, including any minor variations as will be readily apparent. Inaccordance with the present invention the sample is one which mightcontain sphingosine kinase including cell extract, tissue biopsy orpossibly serum, saliva, mucosal secretions, lymph, tissue fluid andrespiratory fluid. The sample is, therefore, generally a biologicalsample comprising biological fluid but also extends to fermentationfluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody havingspecificity for the sphingosine kinase or antigenic parts thereof, iseither covalently or passively bound to a solid surface. The solidsurface is typically glass or a polymer, the most commonly used polymersbeing cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene. The solid supports may be in the form of tubes, beads,discs of microplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes) and under suitable conditions (e.g. 25° C.) to allotsbinding of any sub unit present in the antibody. Following theincubation period, the antibody sub unit solid phase is washed and driedand incubated with a second antibody specific for a portion of thehapten. The second antibody is linked to a reporter molecule which isused to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in thebiological sample and then exposing the immobilized target to specificantibody which may or may not be labelled with a reponer molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound target may be detectable by direct labellingwith the antibody. Alternatively, a second labelled antibody, specificto the first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase and alkaline phosphatase, amongst others.The substrates to be used with the specific enzymes are generally chosenfor the production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labelledantibody is added to the first antibody hapten complex, allowed to bind,and then the excess reagent is washed away. A solution containing theappropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of hapten which was present in the sample.“Reporter molecule” also extends to use of cell agglutination orinhibition of agglutination such as red blood cells on latex beads, andthe like.

Alternately, fluorescent compounds, such as fluorecein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorocrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescene and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

The present invention also contemplates genetic assays such as involvingPCR analysis to detect sphingosine kinase or its derivatives.

Further features of the present invention are more fully described inthe following non-limiting examples.

EXAMPLE 1 Purification and Cloning of Human SphingosineKinase—Experimental Procedures Materials

D-erythro-Sphingosine, D-erythro-dihydrosphingosine,DL-thieo-dihydrosphingosine, N,N-dimethylsphingosine,N-acetylsphingosine (C₂-ceramide). S1P and Fumosin B1 were purchasedfrom Biomol Research Laboratories Inc. (Plymouth Meeting, Pa.).Phytosphingosine, L-α-phosphatidic acid. L-α-phosphatidylinositol,L-α-phosphatidylserine, L-α-phosphatidylcholine.L-α-phosphatidyldethanolamine. 1,2-dioctanoyl-sn-glycerol,1,2-dioleoyl-sn-glycerol, ATP, calmodulin, glutathione and bovine serumalbumin (BSA) were from Sigma. N,N,N-trimethylsphingosine and ADP werepurchased from Calbiochem (Band Soden, Germany), [γ³²P]ATP fromGeneworks (Adelaide, South Australia), TNFα from R&D Systems Inc.(Minneapolis, Minn.), and isopropyl-β-D-thiogalactoside (IPTG) fromPromega (Madison, Wis.). Prepacked Mono-Q, Superose 75 and HiTrap-Qcolumns, Q Sepharose fast flow, calmodulin Sepharose 4B, glutathioneSepharose 4B, thrombin and gel filtration molecular mass proteinstandards were from Amersham Pharmacia Biotech. SDS-PAGE molecular massprotein standards, silver stain plus kit, Coomassie Brilliant Blue R250and Coomassie protein reagent were from Bio-Rad. Centricon concentratorswere purchased from Amicon Inc. (Beverly, Mass.) and BCA protein reagentwas from Pierce Chemical Company (Rockford, Ill.).

Sphingosine Kinase Enzyme Assay

Sphingosine kinase activity was routinely determined usingD-erythro-sphingosine and [γ³²P]ATP as substrates, essentially aspreviously described (Olivera et al., 1998) with some modifications.Briefly, assays were performed by incubating samples at 37° C. for 30min with sphingosine (100 μM stock dissolved in 5% Triton X-100) and[γ-³²P]ATP (1 mM; 10 μCi/ml) in assay buffer containing 100 mM Tris/HCl(pH 7.4), 10 mM MgCl₂ 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mMEGTA, 1 mM Na₃VO₄, 15 mM NaF, 0.5 mM 4-deoxypyridoxine in a total volumeof 100 μl. Reactions were terminated and sphingosine-1-phosphateextracted by the addition of 700 μl of chloroform/methanol/HCl(100:200:1. v/v), followed by vigorous mixing, addition of 200 μp ofchloroform and 200 μl of 2 M KCl, and phase separation bycentrifugation. The labeled S1P in the organic phase was isolated by TLCon Silica Gel 60 with 1-butanol/ethanol/acetic acid/water (8:2:1:2, v/v)and quantitated by phosphorimager (Molecular Dynamics. Sunnyvale,Calif.). One unit (U) of activity is defined as 1pmol of S1P formed perminute.

Purification of Sphingosine Kiniase From Human Placenta

Sphingosine kinase was purified from 1240 g of human placenta (4placentas), with all steps performed at 4° C. The placentas were diced,washed in buffer A (25 mM Tris/HCl buffer, pH 7.4 containing 10% (v/v)glycerol, 0.05% Triton X-100 and 1 mM dithiothreitol), transfered to 1.5L of fresh buffer A containing a protease inhibitor cocktail (Complete™;Boehringer Mannheim) (buffer B), and minced in a Waring blender. Theresultant homogenate was stored on ice for 30 min to enhance enzymeextraction, and the soluble fraction of the homogenate then isolated bycentrifugation at 17 000 g for 60 min. This preparation was thenfractionated by (NH₄)₂SO₄ precipitation by the addition of solid(NH⁴)²SO⁴ at pH 7.4 and collection of the precipitated proteins bycentrifugation (17 000 g, 30 min). The 25-35%-saturated (NH₄)₂SO₄fraction was then redissolved in a buffer B, desalted by extensivedialysis against this same buffer, and centrifuged (17 000 g, 30 min) toremove insoluble material. All subsequent chomatographic steps wereperformed using a FPLC system (Pharmacia Biotech) at 4° C.

The dialysed (NH₄)₂SO₄ fraction was applied to a Q-Sepharose fast flowcolumn (50 mm diameter, 250 ml bed volume) pre-equilibrated with bufferA at a flow rate of 7 ml/min. Sphingosine kinase activity was elutedwith a NaCl gradient of 0 to 1 M in buffer A and collected in 10 mlfractions. Fractions containing highest SK1 activity were then combined,and CaCl₂ and NaCl added to give final concentrations of 4 mM and 250mM, respectively. This pooled extract was then applied to acalmodulin-Sepharose 4B column (16 mm diameter, 10 ml bed volume),pre-equilibrated with buffer A containing 2 mM CaCl₂, at a flow rate of1 ml/min. The column was then washed with several column volumes ofequilibration buffer, followed by buffer A containing 4 mM EGTA, andthen SB eluted with buffer A containing 4 mM EGTA and 1 M NaCl. Thefractions containing highest sphingosine kinase activity were pooled,desalted on a Sephadex G-25 column, and applied at a flow rate of 1ml/min to a Mono-Q column (5 mm diameter 1 ml bed volume)pre-equilibrated with buffer A. Sphingosine kinase activity was elutedwith a NaCl gradient to 0 to 1M in buffer A. NaCl (to 500 mM) wasimmediately added to the fractions (1 ml) collected to stabilise enzymeactivity. The Mono-Q fractions containing highest sphingosine kinaseactivity were combined and desalted on a Sephadex G-25 column. ATP andMgCl₂ were then added to the pooled fractions to a final concentrationsof 1 mM and 5 mM, respectively, before reapplication at a flow rate of 1ml/min to the Mono-Q column pre-equilibrated with buffer A containing 1mM ATP and 5 mM MgCl₂. Sphingosine kinase activity was eluted with aNaCl gradient of 0 to 1M in the equilibration buffer. Again, NaCl (to500 mM) was immediately added to the fractions (1 ml) collected tostabilise enzyme activity. The ATP-Mono-Q fractions containing highestsphingosine kinase activity were pooled and concentrated 10-fold to afinal volume of 200 μl in a Centricon- 10 concentrator and applied at aflow rate of 0.4 ml/min to a Superdex 75 column (10 mm diameter, 20 mlbed volume) pre-equilibrated with buffer A containing 500 mM NaCl.Sphingosine kinase activity was eluted with the same buffer and 0.4 mlfractions collected. The molecular mass of the enzyme was estimated fromthis column by comparison to the elution volumes of ribonuclease A,chymotrypsinogen A, ovalbumin and BSA.

Cloning of Human Sphingosine Kinase

The human sphingosine kinase (hSK) was amplified from a HUVEC λ Zap cDNAlibrary using PCR primers derived from human EST sequences (GenBankaccess-ion numbers D31133, W63556, AA026479, AA232791, AA081152,AI769914 and AI769914) aligned to the murine sphingosine kinases(Olivera et al., 1998). These primers, spanning a central SacII site(P1, 5′-CGGAATTCCCAGTCGGCCGCGGTA-3′ [SEQ ID NO:3] and P2,5′-TAGAATTCTACCGCGGCCGACTGGCT-3′ [SEQ ID NO:4]), were used incombination with T3 and T7 primers to generate two overlapping PCRproducts of 669 bp and 550 bp that represented the 5′ and 3′ ends ofhSK, respectively. These two PCR products were then separately clonedinto pGEM4Z. A 584 bp SacII fragment from the 5′ hSK PCR clone was thensub-cloned in the correct orientation into the SacII site of the 3′ hSKPCR clone, to generate a 1130 bp partial hSK cDNA clone. A full lengthclone encoding hSK was then generated by sub-cloning a 120 bp EcoRI/StuIfragment from the 669 bp 5′ hSK clone into this pGEM4Z-1130 bp clonedigested with EcoRI/StuI. Sequencing the cDNA clone in both directionsverified the integrity of the hSK cDNA sequence.

For mammalian cell expression the hSK cDNA was FLAG epitope tagged atthe 3′-end by PFU polymerase PCR with oligonucleotide primers T7 and5′-TAGAATTCACTTGTCATCGTCGTCCTTGTAGTCTAAGGGCTCTTCTGGCGGT-3′ [SEQ IDNO:5]. This FLAG-tagged hSK cDNA was then cloned into pcDNA3 bydigestion with EcoRI. The orientation was determined by restrictionanalysis and sequencing verified the integrity of the hSK-FLAG cDNAsequence. For bacterial expression, the full length hSK cDNA wassub-cloned into pGEX4T2. The pGEM4Z-hSK clone was digested with BamHIand blunted with 3U PFU polymerase in 1×PFU buffer, 50 μM dNTP's at 72°C. for 30 minutes. The 1163 bp hSK cDNA was gel purified followingdigestion with SalI and the blunt/SalI fragment was then ligated topGEX4T2 SmaI/XhoI.

Sphingosine Kinase Amino Acid Sequence Analysis

The human sphingosine kinase amino acid sequence was searched againstnon-redundant amino acid and nucleotide sequence databases at theAustralian National Genome Information Service using the blastp andtblastn algorithms (Altschul et al., 1990).

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were isolated aspreviously described (Wall et al., 1978) and cultured on gelatin-coatedculture flasks in medium M199 with Earle's salts supplemented with 20%fetal calf serum, 25 μg/ml endothelial growth supplement (CollaborativeResearch) and 25 μg/ml heparin. The cells were passaged three times andgrown to 80% confluency before treatment and harvesting. Human embryonickidney cells (HEK293, ATCC CRL-1573) cells were cultured on Dulbecco'smodified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine,0.2% (w/v) sodium bicarbonate, penicillin (1.2 mg/ml), and gentamycin(1.6 mg/ml). HEK293 cells were transiently transfected using the calciumphosphate precipitation method (Graham & van der Eb, 1973). Treatment ofHUVEC and HEK293 cells with TNFα (1 ng/ml) was performed as previouslydescribed (Xia et al., 1998).

EXAMPLE 2 Expression and Isolation of Recombinant Human SphingosineKinase from E. Coli—Experimental Procedure

The full length SPHK cDNA cloned into pGEX4T2 was transformed into E.coli BL21. Overnight cultures (100 ml) of transformed isolates weregrown with shaking (200 rpm) at 30° C. in Superbroth (20 g/L glucose, 35g/L tryptone, 20 g/L yeast extract, 5 g/L NaCl, pH 7.5) mediumcontaining ampicillin (100 mg/L). The cultures were diluted 1:20 infresh medium and grown at 30° C. with shaking to an OD₆₀₀ of 0.6-0.7.Expression of the glutathione-s-transferase (GST)-coupled sphingosinekinase (GST-SK) was then induced by addition of 0.1 mMisopropyl-β-D-thiogalactoside and further incubation of the cultures at30° C. for 3 h. After this time the bacterial cells were then harvestedby centrifugation at 6,000 g for 20 min at 4° C. and resuspended in 20ml of buffer B containing 250 mM NaCl. the cells were then lysed withlysozyme at a final concentration of 0.3 mg/ml for 15 min at 25° C.followed by sonication, consisting of three cycles of 20 s ultrasonicpulses followed by one minute cooling. The lysate was then clarified bycentrifugation at 50,000 g for 45 min at 4° C., followed by filtratingthrough 0.22 μm filters. To be filtered supernatant was then incubatedwith 0.2 volumes of 50% (w/v) glutathione-Sepharose 4B (Pharmacia) thatwas washed and pro-equilibrated with buffer B, for 60 min at 4° C. withconstant mixing. After this time the mixture was poured into a glasschromatography column (10 mm diam.) and the beads (with bound GST-SK)washed with 10 column volumes of buffer B at 4° C. The GST-SK was theneluted from the column in 10 ml of buffer B containing 10 mM reducedglutathione. Cleavage of the GST away from sphingosine kinase was thenperformed by incubation with 20 μg (30 N.I.H. units) thrombin(Pharmacia) for 3 h at 25° C. The released sphingosine kinase was thenpurified by application of the cleavage mix to a calmodulin-Sepharosecolumn and then a Mono-Q anion exchange column for the purification ofthe sphingosine kinase from human placenta. These columns resulted inpurification of the recombinant sphingosine kinase to homogeneity.

EXAMPLE 3 Characterization of Sphingosine Kinases—ExperimentalProcedures

The effect of pH on the activity of the isolated sphingosine kinases wasdetermined over the pH range 4.0 to 11.0 in 50 mM buffers (sodiumacetate, pH 4.0-5.0; Mes, pH 6.0-7.0; Hepes, pH 7.0-8.2; Tris/HCl, pH8.2-10.0: Caps, pH 10.0-11.0) at 37° C. pH stability was determined byassaying the residual activity after pre-incubation of the enzymes inthe same buffers for 5 h at 4° C. Similarly, thermal stabilities weredetermined by assaying the residual activity after pre-incubation of theenzymes at various temperatures (4-80° C.) for 30 min at pH 7.4 (50 mMTris/HCl containing 10% glycerol, 0.5 M NaCl and 0.05% Triton X-100).Substrate kinetics were analysed using Michaelis-Menten kinetics with aweighted non-linear regression program (Easterby, 1996). Sincesphingosine and its analogues were added to the enzyme assays in mixedmicelles with Triton X-100, where they exibit surface dilution kinetics(Buehrer and Bell. 1992), all K, and KC values obtained for thesemolecules were expressed as mol % of Triton X-100, rather than as bulksolution concentrations. For assays to determine the effect ofcalcium/calmodulin on sphingosine kinase activity, calcium andcalmodulin were added to the standard assay mixtures containing 20 nM ofisolated sphingosine kinase at final concentrations of 4 mM and 0.6 μM,respectively.

EXAMPLE 4 Other Analytical Methods

Protein was determined using either the Coomassie Brilliant Blue(Bradford-et al., 1976) or Bicinchoninic acid (Smith et al., 1985)reagents using BSA as standard. In some cases protein estimations wereperformed after concentration and removal of detergent by precipitation(Wessel and Flügge, 1984) to increase the sensitivity and accuracy ofthe determinations. SDS-PAGE was performed according to the method ofLaemmli (1970) using 12% acrylamide gels. Protein bands on gels werevisualised with either Coomassie Brilliant Blue R250 or silver staining.Molecular mass was estimated by comparison to the electophoriticmobility of myosin, β-galactosidase, BSA, ovalburninm, carbonicanhydrase soybean trypsin inhibitor, lysozyme and aprotinin.

EXAMPLE 5 Purification of Human Sphingosine Kinase—Results

Just over half of the total sphingosine kinase activity in humanplacenta was present in the cytosol after tissue homogenisation (Table3). The purification of sphingosine kinase from human placenta issummarised in Table 4. The soluble fraction from the homogenate of fourhuman placentae was initially subjected to ammonium sulphateprecipitation. This resulted in a remarkably good purification ofsphingosine kinase (33-fold), which precipitated in the 25-35% saturatedammonium sulphate fraction. For anion exchange chromatography theammonium sulphate fraction was rapidly desalted through the use ofSephadex G25 column and the desalted fraction loaded immediately onto aQ Sepharose FF column. The speed of this desalting step appearedcritical since the sphingosine kinase activity appeared very unstable atNaCl concentrations below about 0.2 M, meaning slower desalting steps,such as dialysis, resulted in substantial losses of enzyme activity.Application of a NaCl gradient of 0 to 1 M to the Q Sepharose FF columnresulted in two peaks of sphingosine kinase activity, eluting atapproximately 0.15 M and 0.6 M NaCl, and designated SK1 and SK2,respectively (FIG. 2). For this study SK1 was selected for furtherpurification due to its greater abundance and stability; SK2 activityappeared very unstable and, unlike SK1, could not be stabilised by theaddition of 0.5M NaCl, 10% glycerol and 0.05% Triton X-100. SK1 was alsochosen since it appeared to be the isoform present in HUVEC, asdiscussed later.

Fractions from Q Sepharose FF column containing SK1 were then affinitypurified by application to a calmodulin-Sepharose 4B column in thepresence of 4 mM CaCl₂, and elution of SK1 performed with EGTA and NaCl,resulting in further substantial purification (38-fold) and high enzymeyields. SK1 could not be eluted from the calmodulin Sepharose 4B columnwith EGTA alone, indicating an unusual association of the enzyme withthis affinity matrix. The active fractions that eluted from thecalmodulin Sepharose 4B column were then desalted and applied to twosubsequent steps of analystical anion exchange chromatography on a MonoQ column, with the second step performed in the presence of 1 mM ATP and5 mM MgCl₂ (FIG. 3). The active fractions resulting from these anionexchange steps were then applied to gel filtration chromatography with aSuperdex 75 column as a final purification step. SK1 eluted from thiscolumn as a single peak with a molecule mass corresponding to 44 kDa.Analysis of the active fraction from this final column by SDS-PAGE withsilver staining (FIG. 4) revealed a single band of molecular mass 45kDa, indicating a homogenous protein that has been purified over amillion-fold from the original placenta extract with remarkably goodyield of 7% of the original sphingosine kinase activity (Table 4). Thisis the first sphingosine kinase to be purified to homogeneity from ahuman source.

EXAMPLE 6 Sphingosine Kinase Isoforms in HUVEC—Results

Since two sphingosine kinase activities were identified in humanplacenta (FIG. 2) we examined the multiplicity of this enzyme activityin HUVEC by the use of preparative anion exchange columns. In contrastto other human tissues and cells, application of HUVEC extracts to thesecolumns resulted in the appearance of only a single sphingosine kinasepeak that eluted at the same point as the human placenta SK1 (FIG. 5). 7Similarly, only a single sphingosine kinase peak eluted afterapplication of HUVEC extracts in which sphingosine kinase activity hadbeen stimulated by 10 min treatment of HUVEC with TNFα (Xia el al.,1999). These results would indicate that SK1, the human placentasphingosine kinase isolated in this study, is probably the main isoformfound in HUVEC, and that TNFα treatment results in an increase in theactivity of this enzyme, rather than the activation of another,otherwise latent, isoform.

EXAMPLE 7 Cloning of Human Sphingosine Kinase and Transient Expressionin HEK293 Cells—Results

A human sphingosine kinase cDNA was generated from a HUVEC λ Zap libraryusing primers designed from human ESTs aligned with the published murinesphingosine kinase sequence (Kohama et al., 1998). The cloning strategyis shown in FIG. 6 (prov). The cDNA has an apparent open reading framecoding for 384 amino acids (FIG. 7), it should be noted that thesequence lacked a recognisable Kozak consensus motif raising thepossibility that the actual initiation sequence may not be included inthis cDNA. The sphingosine kinase cDNA encodes for a protein (hSK) witha predicted isoelectric point of 6.64 and a molecular mass of 42,550kDa, consistent with the molecular mass determined for the purifiedhuman placenta sphingosine kinase (SK1). Subcloning into pcDNA3 andtransient expression of hSK in HEK293 cells resulted in a 3200-foldincrease in sphingosine kinase activity in these cells (FIG. 8),compared with untransfected HEK293 cells or HEK293 cells transfectedwith empty vector, indicating that the generated hSK cDNA encodes agenuine sphingosine kinase. Interestingly, although hSK-transfectedHEK293 cells had 3200-fold higher levels of sphingosine kinase activity,treatment of these cells with TNFα resulted in a rapid (10 min) increasein sphingosine kinase activity by a similar proportion (approximately2-fold) to that seen in untransfected HEK293 cells (FIG. 8) (Xia et al.,1998). This indicates the high levels of over-expressed sphingosinekinase are not saturating the TNFα mediated activation mechanism inthese cells.

EXAMPLE 8 Human Sphingosine Kinase Analysis—Results

A search of the database shown hSK has a high amino acid sequencesimilarity (28 to 36% identity) to two recently identified Saccharomycescerevisiae sphirigosine kinases (Nagiec et al., 1998) and several otherESTs encoding putative sphingosine kinase proteins fromSchizosaccharomyces pombe, Caenorhabditis elegans and Arabidopisthaliana. Multiple sequence alignment of hSK with these homologues (FIG.9) revealed several regions of highly conserved amino acids throuaht theprotein, but particularly towards the N-terminus.

A search of the domain structures of hSK sequence revealed threecalcium/calmodulin binding motifs (Rhoads & Friedberg, 1997), one of the1-8 14 type A ([FILVW]xxx{FAILVW]xx[FAILVW]xxxxx{FILVW] (SEQ ID NO:6)with net charge of +3 to +6) spanning residues 290 to 303, and two ofthe 1-8 14 type B ([FILVW]xxxxx{FAILVW]xxxxx[FILVW] (SEQ ID NO:7) withnet charge of +2 to +4) that overlap between residues 134 to 153.Further analysis of the hSK sequence revealed a possibleN-myristoylation site close to the N-terminus (at Gly⁵) that may beapplicable if the protein is subject to proteolytic cleavage. Alsoidentified were one putative casein kinase II (CKII) phosphorylationsite (at Ser¹³⁰) and four putative PKC phosphorylation sites (at Thr⁵⁴,Ser¹⁸⁰, Thr²⁰⁵ and Ser³⁷¹). (FIG. 7). These putative phosphorylationsites are also found in both murine sphingosine kinase isoforms,although the mouse enzymes also display six more possiblephosphorylation sites; four for PKC, and one each for CKII and proteinkinase A, that do not occur in hSK.

A search of signalling domain sequences using the SMART search tool(Schultz et al., 1998; Ponting et al., 1999) revealed similarity inresidues 16 to 153 of hSK to the putative diacylglycerol kinase (DGK)catalytic domain. hSK showed an overall 36% identity to the consensussequence of the DGK catalytic domain family, and possessed 17 of the 24very highly conserved amino acids of this domain. hSK, however, showedno homology with the proposed ATP binding motif of this domain(GxGxxGX_(n)K) (SEQ ID NO:8), although it should be noted that theapplicability of this protein kinase ATP-binding site motif (Hanks etal., 1988) to DGKs remains contentious (Schaap et al., 1994); Sakane etal., 1996; Masai et al., 1993). Further sequence analysis of the humansphingosine kinases also failed to find regions showing any markedsimilarity to the proposed nucleotide-binding motifs found in otherprotein families (Saraste et al., 1990; Walker et al., 1982). Apart fromthe similarity to the DGK catalytic domain, hSK shows no similarity toother lipid binding enzymes, and does not appear to have anyrecognizable lipid binding domains, like PKC C2 or pleckstrin homologydomains. There are also no other obvious regulatory domains, with thepossible exception of a proline-rich region at the C-terminus which hassome similarity to SH3 binding domains (Ren et al., 1993; Yu et al.,1994).

EXAMPLE 9 Expression in E. Coli and Isolation of Recombinant SphingosineKinase—Results

The hSK cDNA was subcloned into the pGEX 4T-2 plasmid and hSK expressedas a glutathione s-transferase (GST) fusion protein in E. coli BL21 byIPTG induction (FIG. 10). After the GST-hSK fusion protein was partiallypurified using glutathione Sepharose 4B, and the GST removed by thrombincleavage, the hSK was further purified by subsequent elutions fromcalmodulin Sepharose and Mono-Q anion exchange columns. This resulted inhigh recovery of sphingosine kinase activity (greater than 70% of theoriginally induced activity), and an electrophoretically puresphingosine kinase (FIG. 10). However, only low protein yields of therecombinant enzyme could be obtained since a large proportion of theIPTG induced, thrombin-cleaved hSK protein did not bind to thecalmodulin Sepharose column (FIG. 11). This non-binding form of hSK hadno demonstrable catalytic activity, suggesting that it was incorrectlyfolded.

EXAMPLE 10 Post-translational Modification Requirement for SphingosineKinase Fuctional Activity

To determine if post-translational modifications are required foractivity of the native sphingosine kinase, the native molecule has beencompared to the recombinant enzyme produced in E. coli where suchmodifications would not occur. Specifically, the enzymes have beenexamined for differences in substrate affinity and accessibility. Thepremise for this study was that post-translational modifications maycause conformational changes in the structure of sphingosine kinasewhich may result in detectable changes in the physico-chemical orcatalytic properties of the enzyme. In summary, it was determined thatrecombinantly produced sphingosine kinase retains its functionalactivity even in the absence of post-translational modification.

METHODS Substrate Specificity of the Native and Recombinant SphingosinieKiniases

Relative rates of phosphorylation of sphingosine by the native andrecombinant sphingosine kinases were arbitrarily set at 100% andcorrespond to 2.65 kU and 7.43 kU of the native and recombinantsphingosine kinases, respective. The substrates examined were added to afinal concentration of 100 μM in 0.25% (w/v) Triton X-100, and assayedunder the standard assay conditions outlined earlier.

Substrate and Inhibitor Kinetics of the Native and RecombinantSphingosine Kinases

Substrate kinetics were determined by supplying substrates over theconcentration range of 0.5 to 200 μM for sphingosine analogues, and 5 to1000 μM for ATP. Inhibition kinetics were determined by the use ofinhibitors over a concentration range of 2 to 50 μM (Table 5). In bothcases the data were analysed by non-linear regression.

Thermal Stabilities of the Native and Recombinant Sphingosine Kinases

Thermal stabilities of the native and recombinant sphingosine kinaseswere determined by assaying the residual activity remaining afterpreincubation of the enzymes at various temperatures (4 to 80° C.) for30 min at pH 7.4 (50 mM Tris/HCl containing 10% glycerol, 0.5M NaCl and0.05% Triton X-100). The original activities of the native andrecombinant sphingosine kinases were arbitrarily set at 100% andcorrespond to 2.65 kU and 7.43 kU, respectively.

pH Stabilities of the Native and Recombinant Sphingosine Kinases

pH stabilities of the native and recombinant sphingosine kinases weredetermined by assaying the residual activity remaining afterpreincubation of the enzymes at various pH's at 4° C. for 5 hr. Theoriginal activities of the native and recombinant sphingosine kinaseswere arbitrarily set at 100% and correspond to 2.65 kU and 7.43 kU,respectively.

The Effect of pH on Activity of the Native and Recombiant SphingosineKinases

The effect of pH on the activity of the native and recombinantsphingosine kinases were determined by assaying the activity over the pHrange of 4 to 1 in 50 mM buffers (sodium acetate, pH 4.0-5.0; Mes, pH6.0-7.0; Hepes, pH 7.0-8.2; Tris, pH 8.2-10.0; Caps. pH 10.0-11.0). Themaximum activities of the native and recombinant sphingosine kinaseswere arbitrarily set at 100% and correspond to 2.65 kU and 7.43 kU,respectively.

Effect of Metal Ions on the Activity of the Native and RecombinantSphingosine Kinases

The effect of metal ions on the activity of the native and recombinantsphingosine kinases were determined by assaying the activity understandard conditions in the presence of various metal ions or EDTA at 10mM. The maximum activities of the native and recombinant sphingosinekinases were arbitrarily set at 100% and correspond to 2.65 kU and 7.43kU, respectively.

Effect of Phospholipids on the Activity of the Native and RecombinantSphingosine Kinases

The effect of various phospolipids on the activity of the native andrecombinant sphingosine kinases were determined by assaying the activityunder standard conditions in the presence of these phospholipids at 10mol% of Triton X-100. The activities of the native and recombinantsphingosine kinases in the absence of phospholipids were arbitrarily setat 100% and correspond to 2.65 kU and 7.43 kU, respectively. PC,phosphatidylcholine; PS phosphatidylserine; PE,phosphatidyldethanolamine; PI, phosphatidylinositol.

RESULTS

Maximum activity of the native and recombinant sphingosine kinases wereobserved at pH 7.4, with both enzymes showing greater than 60% ofmaximum activity in the pH range 6.8 to 7.4 (FIG. 12). Both sphingosinekinases retained more than 90% of the original activity after 5 hincubation at 4° C. in the pH range 6 to 7.8 (FIG. 12), and at pH 7.4 inthe presence of 10% glycerol, 0.5M NaCl and 0.05% Triton X-100, bothenzymes were stable for 30 min at temperatures up to 37° C. (FIG. 12).The enzymes were much less stable in buffers lacking glycerol. NaCl andTriton X-100 (data not shown), consistent with previous observations ofthe bovine brain and rat kidney sphingosine kinases (Louie et al., 1976;Olivera et al., 1998). Both human sphingosine kinases showed arequirment for divalent metal ions since the presence of EDTA in assayselimated activity (FIG. 12). Like other sphingosine kinases examined(Louie et al., 1976; Buelirer & Bell. 1992; 1993; Olivera et al., 1998;Nagiec et al., 1998), both human enzymes showed highest activity withMg²⁺, somewhat lower activity with Mn²⁻, and only very low activity withCa²⁺. Other divalent metal ions examined, including ZN²⁻, Cu²⁻ and Fe²⁺,did not support sphingosine kinase activity (FIG. 12).

The native and recombinant sphingosine kinases have very similar, andnarrow, substrate specificites (FIG. 13), with both showing greatestactivity with the naturally occurring mammalian substrateD-erythro-sphingosine as well as D-erhthro-dihydrosphingosine. Lowactivity was also detected for both enzymes against phytosphingosine,while a range of other sphingosine derivatives and related moleculeswhere not phosphorylated. These included DL-threo-dihydrosphingosine,N,N-dimethylsphingosine, N,N,N-trimethylsphingosine, N-acetylsphingosine(C₂-ceramide), diacylglycerol (1,2-dioctanoyl-sn-glycerol and1,2-dioleoyl-sn-glycerol), and phosphatidylinositol. Further analysis ofthe human sphingosine kinases with D-erythro-sphingosine as well asD-erhthro-dihydrosphingosine revealed Michaelis-Menten kinetics over theconcentration range used (FIG. 13), with both isolated native andrecombinant sphingosine kinases showing very similar kinetic propertiesand slightly higher affinity for D-erythro-sphingosine as well asD-erhthro-dihydrosphingosine (Table 5). Both enzymes also displayedsimilar kinetics when sphingosine was supplied as a sphingosine-BSAcomplex, although presentation of the substrate in this manner resultedin a lower k_(cat) values for both enzymes (28 s⁻¹ and 39 s⁻¹ for thenative and recombinant sphingosine kinases, respectively) compared toits presentation in Triton X-100 mixed micelles, as used for all theother assays performed in this study. Sphingosine supplied as a BSAcomplex K_(m) values of 16±4 mM and 17±2 mM for the native andrecombinant sphingosine kinases, respectively. Both the native andrecombinant sphingosine kinases had the same affinity for ATP (K_(m) ofapprox. 80 mM (Table 5).

Both the native and recombinant sphinqosine kinase were inhibited byDL-threo-dihydrosplhingosine, N,N-dimethylsphingosine andN,N,N-trimethylsphingosine (FIG. 13). with all three of these moleculesdisplaying competitive inhibition with respect to sphingosine. Althoughthe inhibition constants for these molecules were quite similar,N,N,N-trimethylsphingosine gave slightly more efficient inhibition thanDL-threo-dihydrosphingosine, which was a marginally more efficientinhibitor than N,N-dimethylsphingosine (Table 5).

ADP also showed weak competitive inhibition, with respect to ATP (Table5). In all cases remarkably similar inhibition constants were observedfor the native and recombinant sphingosine kinases (Table 5). Noinhibition was seen with N-acetylsphingosine or Fumosin B1, a ceramidesynthase inhibitor.

The effect of calcium/calmodulin on sphingosine kinase activity wasexamined. Under the assay conditions used, calcium/calmodulin had noeffect on the activity of either the native or recombinant humansphingosine kinases (data not shown). While this result indicates a lackof sphingosine kinase activity regulation by calmodulin, the possibilityremains that calmodulin may be involved in some other function withsphingosine kinase, such as regulating ists subcellular localisation.

Stimulation of sphingosine kinase activity by acidic phospholipids wasexamined. The addition of the neutral phospholipids phosphatidylcholineand phosphatidyldethanolamine to the enzyme assay mixture did not resultin any detectable differences in human sphingosine kinase activity,however, marked increases in activity (1.6 to 2-fold) were observed withthe acidic phospholipids phosphatidylserine, phosphatidylinositol andphosphatidic acid (FIG. 14). Both the native and recombinant sphingosinekinases were activated in a similar manner by these acidicphospholipids, with kinetic analyses revealing that all threephospholipids caused an increase in the enzymes K_(cat), while the K_(m)values remained unchanged.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 3 Comparison of sphingosine kinase activity in human placenta withvarious animal tissues Fresh animal tissues were washed and homogenizedas described for the human placenta. The proportion of cytosolicsphingosine kinase activity was determined by comparison of the totalactivity in the homogenate to that from the ultracentrifugationsupernatant (100,000 × g. 60 min). The specific activity of sphingosinekinase is expressed as pmol of SIP formed per minute (U) per g tissue.Specific activity Cytosolic (U/g tissue) (%) Human placenta 13 51 Ratkidney 28 63 Rat liver 19 37 Rat brain 13 52 Sheep kidney 38 58 Sheepliver 17 31 Sheep brain 16 63 Sheep spleen 9 34

TABLE 4 Purification of sphingosine kinase from human placentaSphingosine kinase was purified from 1240 g of fresh human placenta (4placentas). One unit (U) of sphingosine kinase activity is defined as 1pmol of SIP formed from sphingosine and ATP per minute. SpecificActivity Protein Activity Recovery Purification Step (U × 10³) (mg)(U/mg) (%) Fold Soluble fraction of homogenate 7943 123600 58 100Ammonium sulphate (25-35%) 7723 3527 1966 97 33 Q Sepharose anionexchange 4048 1098 4597 63 79 Calmodulin Sepharose 3197 18.23 1.75 × 10⁵40 3.0 × 10³ Mono Q anion exchange 1706 2.921 5.84 × 10⁵ 21.5 1.0 × 10⁴ATP-Mono Q anion exchange 1133 0.419 2.70 × 10⁶ 14.3 4.6 × 10⁴ Superdex75 gel filtration 549 0.008 6.64 × 10⁷ 6.9 1.1 × 10⁶

TABLE 5 Substrate and inhibition kinetics of the native and recombinantsphingosine kinases Substrate kinetics were determined by supplyingsubstrates over the concentration range of 0.5 to 200 μM (0.0125 to 5mol %) for sphingosine analogues. and 5 to 1000 μM for ATP. Inhibitionkinetics were determined by the use of inhibitors over a concentrationrange of 2 to 50 μM (0.05 to 1.25 mol %) for sphingosine analogues and0.1 to 5 mM for ADP. For comparison to previous studies, all K_(m) andK_(i) values for sphingosine and its derivatives are expressed as bothbulk solution concentrations and mol % of Triton X-100, where TritonX-100 was present in all assays at a final concentration of 0.25% (w/v).All kinetic values and standard errors (Duggleby, 1981) were obtainedfrom non-linear regression analysis (Easterby, 1996). Native SKRecombinant SK SUBSTRATE KINETICS: Sphingosine K_(m) (mol %) 0.35 ± 0.050.30 ± 0.07 K_(m) (μM) 14 ± 2  12 ± 3  k_(cat) (S⁻¹) 50 85 k_(cat)/K_(m)(10⁻⁵ s⁻¹ · M⁻¹) 36 71 Dihydrosphingosine K_(m) (mol %) 0.50 ± 0.05 0.48± 0.05 K_(m) (μM) 20 ± 2  19 ± 2  k_(cat) (S⁻¹) 35 76 k_(cat)/K_(m)(10⁻⁵ s⁻¹ · M⁻¹) 18 39 ATP K_(m) (μM) 77 ± 11 86 ± 12 INHIBITORKINETICS: N,N-dimethylsphingosine K_(i) (mol %) 0.20 ± 0.03 0.19 ± 0.02K_(i) (μM) 7.8 ± 1   7.5 ± 1   DL-threo-dihydrosphingosine K_(i) (mol %)0.15 ± 0.02 0.14 ± 0.03 K_(i) (μM) 5.9 ± 1   5.7 ± 1  N,N,N-trimethylsphingosine K_(i) (mol %) 0.12 ± 0.03 0.10 ± 0.02 K_(i)(μM) 4.6 ± 1   3.8 ± 1   ADP K_(i) (mM) 1.1 ± 0.3 0.9 ± 0.2

BIBLIOGRAPHY

Altschul, S. F., Gish. W., Miller, W., Meyer, E. W., and Lipman, D. J.,(1990) J. Mol. Biol. 215:403-410

Alessenko, A. V., (1998) Biochermistry 63:62-68

Bonner et al., (1973) J. Mol. Biol. 81:123

Bradford, M. M., (1976) Anal. Biochem. 72:248-254

Buehrer, B. M., and Bell, R. M., (1992) J. Biol Chem. 267:3154-3159

Buefhrer, B. M., and Bell, R. M., (1993) Adv. Lipid Res. 26:59-67

Buehrer, B. M., Bardes, E. S., and Bell. R. M., (1996) Biochimn.Biophys. Acta 1303:233-242

Culliver, O., Pirianov, G., Kleuser. B., Vanek, P. G., Coso, O. A.,Gutkind, J. S., and Spiegel, S., (1996) Nature 381:800-803

Douillard and Hoffman, Basic facts about hybridomas in Compendium ofImmunology, Vol II ed. Schwartz (1981)

Duggleby, R. G., (1981) Anal Biochem. 110:9-18

Easterby, J. S., (1996) Hyper 1.1s: Hyperbolic Regression Analysis ofEnzyme Kinetic Data. Liverpool University, Liverpool

Graham, F. L., and van der Eb, A. J. (1973) Virology 54:536-539

Hanks, S. K., Quinn, A. M., and Hunter, T., (1988) Science 241:42-52

Igarashi. Y., (1997) J. Biochem. 122:1080-1087

Kohama. T., Olivera, A., Edsall. L., Naiec. M. M., Dickson. R. andSpiegel, S., (1998) J Biol. Chem. 273:23722-23728

Kohler and Milstein., (1975) Nature 256:495-499

Laemmli, U. K., (1970) Nature 227:680-685

Louie, D. D., Kisic. A., and Schroepfer. G. J., (1976) J. Biol. Chem.251:4557-4564

Masai, I., Okazaki, A., Hosoya, T., and Hotta, Y., (1993) Proc. Natl.Acad. Sci. USA 90:11157-11161

Melendez, A., Floto, R. A., Gillooly, D. J., Hamett, M. M. and Allen, J.M., (1998) J. Biol Chem. 273:9393-9402

Meyer zu Heringdorf, D., van Koppen. C. J. and Jakobs, K. H., (1997)FEBS Lett 410:34-38

Nagiec, M. M., Skrzypek, M., Nagiec, E. E., Lester, R. L., and Dickson,R. C., (1998) J Biol. Chem. 273:19437-19442

Olivera, A. and Spiegel, S., (1993) Nature 365:557-560

Olivera, A., Kohama, T., Tu, Z., Milstein, S., and Spiegel, S., (1998) JBiol. Chem. 273:12576-12583

Ponting, C. P., Schultz, J., Milpetz., and Bork, P., (1999) NucleicAcids Res. 27:229-232

Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D., (1993) Science259:1157-1161

Rhoads. A. R., and Friedberg. F., (1997) FASEB J. 11:331-340

Sakane. F., Lai. M., Wada I., Imai. S., and Kohoh. H., (1996) Biochem J.318:583-590

Saraste, M., Sibbald, P. R. and Wittinghofer, A., (1990) TrentdsBiochem. Sci. 15:430-434

Schaap, D., van der Wal, J., and van Blitterswijk, W., (1994) Biochem.J. 304:661-662

Schultz, J., Milpetz, F., Bork, P., and Ponting. C. P., (1998) Proc.Natl, Acad. Sci. USA 95:5857-5864

Smith, P. K., Krohn, R. I., Hermanson. G. T., Mallia, A. K., Gartner, F.H., Provenzano, M.D., Fujimoto, E. K., Goeke, N. M., Olson. B. J., andKienk, D. C., (1985) Anal. Biochem. 150:76-85

Spiegel, S., Culliver, O., Edsall, L., Kohama, T. Menzeleev, R.,Olivera, A., Thomas, D., Tu, Z., Van Brooklyn, J. and Wang, F., (1998).Biochemistry (Mosc) 63:69-73

Walker, J. E., Saraste, M., Runswick. M. J., and Gray, N. J., (1982)EMBO J. 8:945-951

Wall, R. T., Harker, L. A., Quadracci. L. J., and Striker. G. E., (1978)J. Cell. Physiol. 96:203-213

Wessel, D., and Flugge, U. I., (1984) Anal. Biochemn. 138:141-143

Xia, P., Gamble, J. R., Rye, K.-A., Wang, L., Hii. C. S. T., Cockerill,P., Khew-Goodall, Y., Bert, A. G., Barter, P. J., and Vadas, M. A.,(1998) Proc. NatL Acad. Sci. USA 95:14196-14201

Yu, H., Chen, J. K., Feng, S., Dalgamo, D. C., Brauer, A. W., andSchreiber, S. L., (1994) Cell 76:933-945

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 56 <210> SEQ ID NO 1 <211>LENGTH: 1205 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (33)..(1184) <223> OTHERINFORMATION: <400> SEQUENCE: 1 gaattcggca cgaggagccg cgggtcgagg tt atggat cca gcg ggc ggc ccc 53 Met Asp Pro Ala Gly Gly Pro 1 5 cgg ggc gtgctc ccg cgg ccc tgc cgc gtg ctg gtg ctg ctg aac ccg 101 Arg Gly Val LeuPro Arg Pro Cys Arg Val Leu Val Leu Leu Asn Pro 10 15 20 cgc ggc ggc aagggc aag gcc ttg cag ctc ttc cgg agt cac gtg cag 149 Arg Gly Gly Lys GlyLys Ala Leu Gln Leu Phe Arg Ser His Val Gln 25 30 35 ccc ctt ttg gct gaggct gaa atc tcc ttc acg ctg atg ctc act gag 197 Pro Leu Leu Ala Glu AlaGlu Ile Ser Phe Thr Leu Met Leu Thr Glu 40 45 50 55 cgg cgg aac cac gcgcgg gag ctg gtg cgg tcg gag gag ctg ggc cgc 245 Arg Arg Asn His Ala ArgGlu Leu Val Arg Ser Glu Glu Leu Gly Arg 60 65 70 tgg gac gct ctg gtg gtcatg tct gga gac ggg ctg atg cac gag gtg 293 Trp Asp Ala Leu Val Val MetSer Gly Asp Gly 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Phe Leu Arg 185 190 195 ctg gca gcc ttg cgc acttac cgc ggc cga ctg gct tac ctc cct gta 677 Leu Ala Ala Leu Arg Thr TyrArg Gly Arg Leu Ala Tyr Leu Pro Val 200 205 210 215 gga aga gtg ggt tccaag aca cct gcc tcc ccc gtt gtg gtc cag cag 725 Gly Arg Val Gly Ser LysThr Pro Ala Ser Pro Val Val Val Gln Gln 220 225 230 ggc ccg gta gat gcacac ctt gtg cca ctg gag gag cca gtg ccc tct 773 Gly Pro Val Asp Ala HisLeu Val Pro Leu Glu Glu Pro Val Pro Ser 235 240 245 cac tgg aca gtg gtgccc gac gag gac ttt gtg cta gtc ctg gca ctg 821 His Trp Thr Val Val ProAsp Glu Asp Phe Val Leu Val Leu Ala Leu 250 255 260 ctg cac tcg cac ctgggc agt gag atg ttt gct gca ccc atg ggc cgc 869 Leu His Ser His Leu GlySer Glu Met Phe Ala Ala Pro Met Gly Arg 265 270 275 tgt gca gct ggc gtcatg cat ctg ttc tac gtg cgg gcg gga gtg tct 917 Cys Ala Ala Gly Val MetHis Leu Phe Tyr Val Arg Ala Gly Val Ser 280 285 290 295 cgt gcc atg ctgctg cgc ctc ttc ctg gcc atg gag aag ggc agg cat 965 Arg Ala Met Leu LeuArg Leu Phe Leu Ala Met Glu Lys Gly Arg His 300 305 310 atg gag tat gaatgc ccc tac ttg gta tat gtg ccc gtg gtc gcc ttc 1013 Met Glu Tyr Glu CysPro Tyr Leu Val Tyr Val Pro Val Val Ala Phe 315 320 325 cgc ttg gag cccaag gat ggg aaa ggt atg ttt gca gtg gat ggg gaa 1061 Arg Leu Glu Pro LysAsp Gly Lys Gly Met Phe Ala Val Asp Gly Glu 330 335 340 ttg atg gtt agcgag gcc gtg cag ggc cag gtg cac cca aac tac ttc 1109 Leu Met Val Ser GluAla Val Gln Gly Gln Val His Pro Asn Tyr Phe 345 350 355 tgg atg gtc agcggt tgc gtg gag ccc ccg ccc agc tgg aag ccc cag 1157 Trp Met Val Ser GlyCys Val Glu Pro Pro Pro Ser Trp Lys Pro Gln 360 365 370 375 cag atg ccaccg cca gaa gag ccc tta tgatctagag tcgacctgca g 1205 Gln Met Pro Pro ProGlu Glu Pro Leu 380 <210> SEQ ID NO 2 <211> LENGTH: 384 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Asp Pro Ala Gly GlyPro Arg Gly Val Leu Pro Arg Pro Cys Arg 1 5 10 15 Val Leu Val Leu LeuAsn Pro Arg Gly Gly Lys Gly Lys Ala Leu Gln 20 25 30 Leu Phe Arg Ser HisVal Gln Pro Leu Leu Ala Glu Ala 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LOCATION: (25)..(25) <223>OTHER INFORMATION: Xaa is unknown <221> NAME/KEY: misc_feature <222>LOCATION: (26)..(26) <223> OTHER INFORMATION: Xaa is unknown <221>NAME/KEY: misc_feature <222> LOCATION: (27)..(27) <223> OTHERINFORMATION: Xaa is unknown <400> SEQUENCE: 6 Phe Ile Leu Val Trp XaaXaa Xaa Phe Ala Ile Leu Val Trp Xaa Xaa 1 5 10 15 Phe Ala Ile Leu ValTrp Xaa Xaa Xaa Xaa Xaa Phe Ile Leu Val Trp 20 25 30 <210> SEQ ID NO 7<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: unknown <221> NAME/KEY:misc_feature <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: Xaa isunknown <221> NAME/KEY: misc_feature <222> LOCATION: (7)..(7) <223>OTHER INFORMATION: Xaa is unknown <221> NAME/KEY: misc_feature <222>LOCATION: (8)..(8) <223> OTHER INFORMATION: Xaa is unknown <221>NAME/KEY: misc_feature <222> LOCATION: (9)..(9) <223> OTHER INFORMATION:Xaa is unknown <221> NAME/KEY: misc_feature <222> LOCATION: (10)..(10)<223> OTHER INFORMATION: Xaa is unknown <221> NAME/KEY: misc_feature<222> LOCATION: (17)..(17) <223> OTHER INFORMATION: Xaa is unknown <221>NAME/KEY: misc_feature <222> LOCATION: (18)..(18) <223> OTHERINFORMATION: Xaa is unknown <221> NAME/KEY: misc_feature <222> LOCATION:(19)..(19) <223> OTHER INFORMATION: Xaa is unknown <221> NAME/KEY:misc_feature <222> LOCATION: (20)..(20) <223> OTHER INFORMATION: Xaa isunknown <221> NAME/KEY: misc_feature <222> LOCATION: (21)..(21) <223>OTHER INFORMATION: Xaa is unknown <400> SEQUENCE: 7 Phe Ile Leu Val TrpXaa Xaa Xaa Xaa Xaa Phe Ala Ile Leu Val Trp 1 5 10 15 Xaa Xaa Xaa XaaXaa Phe Ile Leu Val Trp 20 25 <210> SEQ ID NO 8 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: unknown <221> NAME/KEY: misc_feature <222> LOCATION:(2)..(2) <223> OTHER INFORMATION: Xaa is unknown <221> NAME/KEY:misc_feature <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa isunknown <221> NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>OTHER INFORMATION: Xaa is unknown <221> NAME/KEY: misc_feature <222>LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa is unknown <400>SEQUENCE: 8 Gly Xaa Gly Xaa Xaa Gly Xaa Lys 1 5 <210> SEQ ID NO 9 <211>LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:9 Met Glu Pro Glu Cys Pro Arg Gly Leu Leu Pro Arg Pro Cys Arg Val 1 5 1015 Leu Val Leu Leu Asn Pro Gln Gly Gly Lys Gly Lys Ala Leu Gln Leu 20 2530 Phe Gln Ser Arg Val Gln Pro Phe Leu Glu Glu Ala Glu Ile Thr 35 40 45<210> SEQ ID NO 10 <211> LENGTH: 52 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 10 Ser Gly Asn Ala Leu Ala Ala Ser Val Asn HisTyr Ala Gly Tyr Glu 1 5 10 15 Gln Val Thr Asn Glu Asp Leu Leu Ile AsnCys Thr Leu Leu Leu Cys 20 25 30 Arg Arg Arg Leu Ser Pro Met Asn Leu LeuSer Leu His Thr Ala Ser 35 40 45 Gly Leu Arg Leu 50 <210> SEQ ID NO 11<211> LENGTH: 69 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 11 Ala Tyr Leu Pro Val Gly Thr Val Ala Ser Lys Arg Pro Ala SerThr 1 5 10 15 Leu Val Gln Lys Gly Pro Val Asp Thr His Leu Val Leu GluGlu Pro 20 25 30 Val Pro Ser His Trp Thr Val Val Pro Glu Gln Asp Phe ValLeu Val 35 40 45 Leu Val Leu Leu His Thr His Leu Ser Ser Glu Leu Phe AlaAla Pro 50 55 60 Met Gly Arg Cys Glu 65 <210> SEQ ID NO 12 <211> LENGTH:53 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 12 AlaGly Val Met His Leu Phe Tyr Val Arg Ala Gly Val Ser Arg Ala 1 5 10 15Ala Leu Leu Arg Leu Phe Leu Ala Met Gln Lys Gly Lys His Met Glu 20 25 30Leu Asp Cys Pro Tyr Leu Val His Val Pro Val Val Ala Phe Arg Leu 35 40 45Glu Pro Arg Ser Gln 50 <210> SEQ ID NO 13 <211> LENGTH: 63 <212> TYPE:PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 13 Phe Lys Leu Ile LeuThr Glu Arg Lys Asn His Ala Arg Glu Leu Val 1 5 10 15 Cys Ala Glu GluLeu Gly His Trp Asp Ala Leu Ala Val Met Ser Gly 20 25 30 Asp Gly Leu MetHis Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp 35 40 45 Trp Glu Thr AlaIle Gln Lys Pro Leu Cys Ser Leu Pro Gly Gly 50 55 60 <210> SEQ ID NO 14<211> LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 14 Tyr Ser Val Leu Ser Leu Ser Trp Gly Phe Val Ala Asp Val AspLeu 1 5 10 15 Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Ile Arg Phe ThrVal Gly 20 25 30 Thr Phe Phe Arg Leu Ala Ser Leu Arg Ile Tyr Gln Gly GlnLeu 35 40 45 <210> SEQ ID NO 15 <211> LENGTH: 46 <212> TYPE: PRT <213>ORGANISM: Mus musculus <400> SEQUENCE: 15 Thr His Leu Val Pro Leu GluGlu Pro Val Pro Ser His Trp Thr Val 1 5 10 15 Val Pro Glu Gln Asp PheVal Leu Val Leu Val Leu Leu His Thr His 20 25 30 Leu Ser Ser Glu Leu PheAla Ala Pro Met Gly Arg Cys Glu 35 40 45 <210> SEQ ID NO 16 <211>LENGTH: 49 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE:16 Arg Gly Val Phe Ser Val Asp Gly Glu Leu Met Val Cys Glu Ala Val 1 510 15 Gln Gly Gln Val His Pro Asn Tyr Leu Trp Met Val Cys Gly Ser Arg 2025 30 Asp Ala Pro Ser Gly Arg Asp Ser Arg Arg Gly Pro Pro Pro Glu Glu 3540 45 Pro <210> SEQ ID NO 17 <211> LENGTH: 54 <212> TYPE: PRT <213>ORGANISM: Mus musculus <400> SEQUENCE: 17 Met Trp Trp Cys Cys Val LeuPhe Val Val Glu Cys Pro Arg Gly Leu 1 5 10 15 Leu Pro Arg Pro Cys ArgVal Leu Val Leu Leu Asn Pro Gln Gly Gly 20 25 30 Lys Gly Lys Ala Leu GlnLeu Phe Gln Ser Arg Val Gln Pro Phe Leu 35 40 45 Glu Glu Ala Glu Ile Thr50 <210> SEQ ID NO 18 <211> LENGTH: 52 <212> TYPE: PRT <213> ORGANISM:Mus musculus <400> SEQUENCE: 18 Ser Gly Asn Ala Leu Ala Ala Ser Val AsnHis Tyr Ala Gly Tyr Glu 1 5 10 15 Gln Val Thr Asn Glu Asp Leu Leu IleAsn Cys Thr Leu Leu Leu Cys 20 25 30 Arg Arg Arg Leu Ser Pro Met Asn LeuLeu Ser Leu His Thr Ala Ser 35 40 45 Gly Leu Arg Leu 50 <210> SEQ ID NO19 <211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 19 Ala Tyr Leu Pro Val Gly Thr Val Ala Ser Lys Arg Pro Ala SerThr 1 5 10 15 Leu Val Gln Lys Gly Pro Val Asp 20 <210> SEQ ID NO 20<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 20 Ala Gly Val Met His Leu Phe Tyr Val Arg Ala Gly Val Ser ArgAla 1 5 10 15 Ala Leu Leu Arg Leu Phe Leu Ala Met Gln Lys Gly Lys HisMet Glu 20 25 30 Leu Asp Cys Pro Tyr Leu Val His Val Pro Val Val Ala PheArg Leu 35 40 45 Glu Pro Arg Ser Gln 50 <210> SEQ ID NO 21 <211> LENGTH:63 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 21 PheLys Leu Ile Leu Thr Glu Arg Lys Asn His Ala Arg Glu Leu Val 1 5 10 15Cys Ala Glu Glu Leu Gly His Trp Asp Ala Leu Ala Val Met Ser Gly 20 25 30Asp Gly Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp 35 40 45Trp Glu Thr Ala Ile Gln Lys Pro Leu Cys Ser Leu Pro Gly Gly 50 55 60<210> SEQ ID NO 22 <211> LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 22 Tyr Ser Val Leu Ser Leu Ser Trp Gly Phe ValAla Asp Val Asp Leu 1 5 10 15 Glu Ser Glu Lys Tyr Arg Arg Leu Gly GluIle Arg Phe Thr Val Gly 20 25 30 Thr Phe Phe Arg Leu Ala Ser Leu Arg IleTyr Gln Gly Gln Leu 35 40 45 <210> SEQ ID NO 23 <211> LENGTH: 46 <212>TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 23 Thr His LeuVal Pro Leu Glu Glu Pro Val Pro Ser His Trp Thr Val 1 5 10 15 Val ProGlu Gln Asp Phe Val Leu Val Leu Val Leu Leu His Thr His 20 25 30 Leu SerSer Glu Leu Phe Ala Ala Pro Met Gly Arg Cys Glu 35 40 45 <210> SEQ ID NO24 <211> LENGTH: 49 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400>SEQUENCE: 24 Arg Gly Val Phe Ser Val Asp Gly Glu Leu Met Val Cys Glu AlaVal 1 5 10 15 Gln Gly Gln Val His Pro Asn Tyr Leu Trp Met Val Cys GlySer Arg 20 25 30 Asp Ala Pro Ser Gly Arg Asp Ser Arg Arg Gly Pro Pro ProGlu Glu 35 40 45 Pro <210> SEQ ID NO 25 <211> LENGTH: 55 <212> TYPE: PRT<213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 25 Asn Ile SerSer Gly Thr Val Glu Glu Ile Leu Glu Lys Ser Tyr Glu 1 5 10 15 Asn SerLys Arg Asn Arg Ser Ile Leu Val Ile Ile Asn Pro His Gly 20 25 30 Gly LysGly Thr Ala Lys Asn Leu Phe Leu Thr Lys Ala Arg Pro Ile 35 40 45 Leu ValGlu Ser Gly Cys Lys 50 55 <210> SEQ ID NO 26 <211> LENGTH: 46 <212>TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 26Ser Gly Asn Ala Met Ser Ile Ser Cys His Trp Thr Asn Asn Pro Ser 1 5 1015 Tyr Ala Ala Leu Cys Leu Val Lys Ser Ile Glu Thr Arg Ile Asp Leu 20 2530 Met Cys Cys Ser Gln Pro Ser Tyr Met Asn Glu Trp Pro Arg 35 40 45 SEQID NO 27 LENGTH: 37 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 27 Glu Asn Lys Asp Lys Asn Lys Gly Cys LeuThr Phe Glu Pro Asn Pro 1 5 10 15 Ser Pro Asn Ser Ser Pro Asp Leu LeuSer Lys Asn Asn Ile Asn Asn 20 25 30 Ser Thr Lys Asp Glu 35 SEQ ID NO 28LENGTH: 51 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae<400> SEQUENCE: 28 Asp Gly Thr Ile Asp Leu Val Ile Thr Asp Ala Arg IlePro Val Thr 1 5 10 15 Arg Met Thr Pro Ile Leu Leu Ser Leu Asp Lys GlySer His Val Leu 20 25 30 Glu Pro Glu Val Ile His Ser Lys Ile Leu Ala TyrLys Ile Ile Pro 35 40 45 Lys Val Glu 50 <210> SEQ ID NO 29 <211> LENGTH:64 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400>SEQUENCE: 29 Ile Glu Ile Ala Tyr Thr Lys Tyr Ala Arg His Ala Ile Asp IleAla 1 5 10 15 Lys Asp Leu Asp Ile Ser Lys Tyr Asp Thr Ile Ala Cys AlaSer Gly 20 25 30 Asp Gly Ile Pro Tyr Glu Val Ile Asn Gly Leu Tyr Arg ArgPro Asp 35 40 45 Arg Val Asp Ala Phe Asn Lys Leu Ala Val Thr Gln Leu ProCys Gly 50 55 60 <210> SEQ ID NO 30 <211> LENGTH: 64 <212> TYPE: PRT<213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 30 Leu Ser PheLeu Ser Gln Thr Tyr Gly Val Ile Ala Glu Ser Asp Ile 1 5 10 15 Asn ThrGlu Phe Ile Arg Trp Met Gly Pro Val Arg Phe Asn Leu Gly 20 25 30 Val AlaPhe Asn Ile Ile Gln Gly Lys Lys Tyr Pro Cys Glu Val Phe 35 40 45 Val LysTyr Ala Ala Lys Ser Lys Lys Glu Leu Lys Val His Phe Leu 50 55 60 <210>SEQ ID NO 31 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM:Saccharomyces cerevisiae <400> SEQUENCE: 31 Leu Ser Pro Asn Phe Leu AsnGlu Asp Asn Phe Lys Leu Lys Tyr Pro 1 5 10 15 Met Thr Glu Pro Val ProArg Asp Trp Glu Lys Met Asp Ser Glu Leu 20 25 30 Thr Asp Asn Leu Thr IlePhe Tyr Thr Gly Lys Met Pro Tyr Ile Ala 35 40 45 Lys Asp Thr Lys Phe PhePro Ala Ala Leu Pro Ala 50 55 60 <210> SEQ ID NO 32 <211> LENGTH: 42<212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE:32 Ser Gly Leu Phe Ser Val Asp Gly Glu Lys Phe Pro Leu Glu Pro Leu 1 510 15 Gln Val Glu Ile Met Pro Met Leu Cys Lys Thr Leu Leu Arg Asn Gly 2025 30 Arg Tyr Ile Asp Thr Glu Phe Glu Ser Met 35 40 <210> SEQ ID NO 33<211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 33 Asp Leu Val Glu Glu Ile Leu Lys Arg SerTyr Lys Asn Thr Arg Arg 1 5 10 15 Asn Lys Ser Ile Phe Val Ile Ile AsnPro Phe Gly Gly Lys Gly Lys 20 25 30 Ala Lys Lys Leu Phe Met Thr Lys AlaLys Pro Leu Leu Leu Ala Ser 35 40 45 Arg Cys Ser 50 <210> SEQ ID NO 34<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 34 Ser Gly Asn Ala Met Ser Val Ser Cys HisTrp Thr Asn Asn Pro Ser 1 5 10 15 Tyr Ser Thr Leu Cys Leu Ile Lys SerIle Glu Thr Arg Ile Asp Leu 20 25 30 Met Cys Cys Ser Gln Pro Ser Tyr AlaArg Glu His Pro Lys 35 40 45 <210> SEQ ID NO 35 <211> LENGTH: 53 <212>TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 35Glu His Lys Asn Lys Gly Ser Leu Glu Phe Gln His Ile Thr Met Asn 1 5 1015 Lys Asp Asn Glu Asp Cys Asp Asn Tyr Asn Tyr Glu Asn Glu Tyr Glu 20 2530 Thr Glu Asn Glu Asp Glu Asp Glu Asp Ala Asp Ala Asp Asp Glu Asp 35 4045 Ser His Leu Ile Ser 50 <210> SEQ ID NO 36 <211> LENGTH: 51 <212>TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 36Asp Gly Thr Met Asp Met Val Ile Thr Asp Ala Arg Thr Ser Leu Thr 1 5 1015 Arg Met Ala Pro Ile Leu Leu Gly Leu Asp Lys Gly Ser His Val Leu 20 2530 Gln Pro Glu Val Leu His Ser Lys Ile Leu Ala Tyr Lys Ile Ile Pro 35 4045 Lys Leu Gly 50 <210> SEQ ID NO 37 <211> LENGTH: 64 <212> TYPE: PRT<213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 37 Ile Glu ValVal Tyr Thr Lys Tyr Pro Gly His Ala Ile Glu Ile Ala 1 5 10 15 Arg GluMet Asp Ile Asp Lys Tyr Asp Thr Ile Ala Cys Ala Ser Gly 20 25 30 Asp GlyIle Pro His Glu Val Ile Asn Gly Leu Tyr Gln Arg Pro Asp 35 40 45 His ValLys Ala Phe Asn Asn Ile Ala Ile Thr Glu Ile Pro Cys Gly 50 55 60 <210>SEQ ID NO 38 <211> LENGTH: 64 <212> TYPE: PRT <213> ORGANISM:Saccharomyces cerevisiae <400> SEQUENCE: 38 Leu Ser Phe Leu Ser Gln ThrTyr Gly Leu Ile Ala Glu Thr Asp Ile 1 5 10 15 Asn Thr Glu Phe Ile ArgTrp Met Gly Pro Ala Arg Phe Glu Leu Gly 20 25 30 Val Ala Phe Asn Ile IleGln Lys Lys Lys Tyr Pro Cys Glu Ile Tyr 35 40 45 Val Lys Tyr Ala Ala LysSer Lys Asn Glu Leu Lys Asn His Tyr Leu 50 55 60 <210> SEQ ID NO 39<211> LENGTH: 65 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 39 Arg Asp Leu Ala Asp Ser Ser Ala Asp GlnIle Lys Glu Glu Asp Phe 1 5 10 15 Lys Ile Lys Tyr Pro Leu Asp Glu GlyIle Pro Ser Asp Trp Glu Arg 20 25 30 Leu Asp Pro Asn Ile Ser Asn Asn LeuGly Ile Phe Tyr Thr Gly Lys 35 40 45 Met Pro Tyr Val Ala Ala Asp Thr LysPhe Phe Pro Ala Ala Leu Pro 50 55 60 Ser 65 <210> SEQ ID NO 40 <211>LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae<400> SEQUENCE: 40 Asn Gly Leu Phe Ser Val Asp Gly Glu Lys Phe Pro LeuGlu Pro Leu 1 5 10 15 Gln Val Glu Ile Met Pro Arg Leu Cys Lys Thr LeuLeu Arg Asn Gly 20 25 30 Arg Tyr Val Asp Thr Asp Phe Asp Ser Met 35 40<210> SEQ ID NO 41 <211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM:Schizosaccharomyces pombe <400> SEQUENCE: 41 Phe Cys Glu Tyr Leu Leu AspVal Ala Tyr Lys Gly Ile Lys Arg Ser 1 5 10 15 Arg Arg Phe Ile Val PheIle Asn Pro His Gly Gly Lys Gly Lys Ala 20 25 30 Lys His Ile Trp Glu SerGlu Ala Glu Pro Val Phe Ser Ser Ala His 35 40 45 Ser Ile 50 <210> SEQ IDNO 42 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM:Schizosaccharomyces pombe <400> SEQUENCE: 42 Ser Gly Asn Ala Phe Ser TyrAsn Ala Thr Gly Gln Leu Lys Pro Ala 1 5 10 15 Leu Thr Ala Leu Glu IleLeu Lys Gly Arg Pro Thr Ser Phe Asp Leu 20 25 30 Met Thr Phe Glu Gln LysGly Lys Lys Ala 35 40 <210> SEQ ID NO 43 <211> LENGTH: 17 <212> TYPE:PRT <213> ORGANISM: Schizosaccharomyces pombe <400> SEQUENCE: 43 Glu LysSer Lys Asn Leu Ala Pro Met Ser Glu Ser Ser Asp Ser Asp 1 5 10 15 Lys<210> SEQ ID NO 44 <211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM:Schizosaccharomyces pombe <400> SEQUENCE: 44 Asp Gly Leu Ile Asp Val ValIle Val Tyr Ser Lys Gln Phe Arg Lys 1 5 10 15 Ser Leu Leu Ser Met PheThr Gln Leu Asp Asn Gly Gly Phe Tyr Tyr 20 25 30 Ser Lys His Leu Asn TyrTyr Lys Val Arg Ser Phe Arg Phe Thr Pro 35 40 45 Val Asn Thr 50 <210>SEQ ID NO 45 <211> LENGTH: 63 <212> TYPE: PRT <213> ORGANISM:Schizosaccharomyces pombe <400> SEQUENCE: 45 Cys Glu Val Val Leu Thr ArgArg Lys Asp His Ala Lys Ser Ile Ala 1 5 10 15 Lys Asn Leu Asp Val GlySer Tyr Asp Gly Ile Leu Ser Val Gly Gly 20 25 30 Asp Gly Leu Phe His GluVal Ile Asn Gly Leu Gly Glu Arg Asp Asp 35 40 45 Tyr Leu Glu Ala Phe LysLeu Pro Val Cys Met Ile Pro Gly Gly 50 55 60 <210> SEQ ID NO 46 <211>LENGTH: 63 <212> TYPE: PRT <213> ORGANISM: Schizosaccharomyces pombe<400> SEQUENCE: 46 Tyr Ser Phe Leu Thr Ala Asn Tyr Gly Ile Ile Ala AspCys Asp Ile 1 5 10 15 Gly Thr Glu Asn Trp Arg Phe Met Gly Glu Asn ArgAla Tyr Leu Gly 20 25 30 Phe Phe Leu Arg Leu Phe Gln Lys Pro Asp Trp LysCys Ser Ile Glu 35 40 45 Met Asp Val Val Ser Ser Asp Arg Thr Glu Ile LysHis Met Tyr 50 55 60 <210> SEQ ID NO 47 <211> LENGTH: 42 <212> TYPE: PRT<213> ORGANISM: Schizosaccharomyces pombe <400> SEQUENCE: 47 Thr Val SerThr Ser Pro Glu Ser His Leu Leu Thr Phe Glu Ile Asn 1 5 10 15 Asp LeuSer Ile Phe Cys Ala Gly Leu Leu Pro Tyr Ile Ala Pro Asp 20 25 30 Ala LysMet Phe Pro Ala Ala Ser Asn Asp 35 40 <210> SEQ ID NO 48 <211> LENGTH:41 <212> TYPE: PRT <213> ORGANISM: Schizosaccharomyces pombe <400>SEQUENCE: 48 Gly Lys Arg His Tyr Phe Ala Leu Asp Gly Glu Ser Tyr Pro LeuGlu 1 5 10 15 Pro Phe Glu Cys Arg Val Ala Pro Lys Leu Gly Thr Thr LeuSer Pro 20 25 30 Val Ala Gly Phe Gln Leu Leu Asp Ile 35 40 <210> SEQ IDNO 49 <211> LENGTH: 55 <212> TYPE: PRT <213> ORGANISM: Caenorhabditiselegans <400> SEQUENCE: 49 Glu Asn Glu Gln Leu Thr Ser Val Ile Leu SerArg Lys Pro Pro Pro 1 5 10 15 Gln Glu Gln Cys Arg Gly Asn Leu Leu ValPhe Ile Asn Pro Asn Ser 20 25 30 Gly Thr Gly Lys Ser Leu Glu Thr Phe AlaAsn Thr Val Gly Pro Lys 35 40 45 Leu Asp Lys Ser Leu Ile Arg 50 55 <210>SEQ ID NO 50 <211> LENGTH: 52 <212> TYPE: PRT <213> ORGANISM:Caenorhabditis elegans <400> SEQUENCE: 50 Ser Gly Asn Gly Leu Leu CysSer Val Leu Ser Lys Tyr Gly Thr Lys 1 5 10 15 Met Asn Glu Lys Ser ValMet Glu Arg Ala Leu Glu Ile Ala Thr Ser 20 25 30 Pro Thr Ala Lys Ala GluSer Val Ala Leu Tyr Ser Val Lys Thr Asp 35 40 45 Asn Gln Ser Tyr 50<210> SEQ ID NO 51 <211> LENGTH: 48 <212> TYPE: PRT <213> ORGANISM:Caenorhabditis elegans <400> SEQUENCE: 51 Thr Tyr Arg Pro Tyr Lys ProLys Gly Phe His Pro Ser Ser Asn Val 1 5 10 15 Phe Ser Val Tyr Glu LysThr Thr Gln Gln Arg Ile Asp Asp Ser Lys 20 25 30 Val Lys Thr Asn Gly SerVal Ser Asp Ser Glu Glu Glu Thr Met Glu 35 40 45 <210> SEQ ID NO 52<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans<400> SEQUENCE: 52 Asp Asn Arg Ile His Leu Ser Tyr Ile Leu Trp Lys AspIle Gly Thr 1 5 10 15 Arg Val Asn Ile Ala Lys Tyr Leu Leu Ala Ile GluHis Glu Thr His 20 25 30 Leu Asp Leu Pro Phe Val Lys His Val Glu Val SerSer Met Lys Leu 35 40 45 Glu Val Ile Ser Glu 50 <210> SEQ ID NO 53 <211>LENGTH: 65 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400>SEQUENCE: 53 Tyr Glu Val Val Val Thr Thr Gly Pro Asn His Ala Arg Asn ValLeu 1 5 10 15 Met Thr Lys Ala Asp Leu Gly Lys Phe Asn Gly Val Leu IleLeu Ser 20 25 30 Gly Asp Gly Leu Val Phe Glu Ala Leu Asn Gly Ile Leu CysArg Glu 35 40 45 Asp Ala Phe Arg Ile Phe Pro Thr Leu Pro Ile Gly Ile ValPro Ser 50 55 60 Gly 65 <210> SEQ ID NO 54 <211> LENGTH: 48 <212> TYPE:PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 54 Ala SerPhe Leu Ser Ile Gly Trp Gly Leu Met Ala Asp Ile Asp Ile 1 5 10 15 AspSer Glu Lys Trp Arg Lys Ser Leu Gly His His Arg Phe Thr Val 20 25 30 MetGly Phe Ile Arg Ser Cys Asn Leu Arg Ser Tyr Lys Gly Arg Leu 35 40 45<210> SEQ ID NO 55 <211> LENGTH: 56 <212> TYPE: PRT <213> ORGANISM:Caenorhabditis elegans <400> SEQUENCE: 55 Thr Lys Phe Gln Asn Trp ThrLeu Pro Asp Ser Asp Glu Thr Leu Ala 1 5 10 15 Val Gly Ser Ser Asp LeuGlu Glu Thr Val Val Ile Glu Asp Asn Phe 20 25 30 Val Asn Ile Tyr Ala ValThr Leu Ser His Ile Ala Ala Asp Gly Pro 35 40 45 Phe Ala Pro Ser Ala LysLeu Glu 50 55 <210> SEQ ID NO 56 <211> LENGTH: 32 <212> TYPE: PRT <213>ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 56 Gly Ser His Val ValLeu Asp Gly Glu Val Val Asp Thr Lys Thr Ile 1 5 10 15 Glu Val Ala SerThr Lys Asn His Ile Ser Val Phe Ser Ser Thr Ala 20 25 30

The claims defining the invention are as follows:
 1. An isolatedpolynucleotide encoding a sphingosine kinase, the polyinucleotidecomprising (1) the sequence of SEQ ID NO:1, (2) a sequence at least 90%identical to SEQ ID NO:1, (3) a nucleotide sequence that hybridizes tothe nucleotide sequence of (1) or (2) under high stringency conditionsof about 65° C. and about 50% v/v formamide and about 0.15M salt, (4) anucleotide sequence encoding a polypeptide having the sequence of SEQ IDNO:2, or (5) a nucleotide sequence complementary to the nucleotidesequence of any one of (1) to (4).
 2. An isolated polynucleotideaccording to claim 1, the polynucleotide comprising a sequence at least95% identical to SEQ ID NO:1.
 3. An isolated polynucleotide sequencethat hybridizes to the nucleotide sequence of claim 2 stringencyconditions of about 65° C. and about 50% v/v formamide and about 0.15Msalt.
 4. An isolated polynucleotide according to claim 1, thepolynucleotide encoding a polypeptide having the sequence of SEQ IDNO:2.
 5. An isolated polynucleotide according to claim 1, thepolynucleotide comprising the sequence of SEQ ID NO:1.
 6. An expressionsystem comprising a polynucleotide according to claim
 1. 7. Anexpression system according to claim 6 capable of producing asphingosine kinase comprising the polypeptide sequence of SEQ ID NO:2when the expression system is in a compatible host cell.
 8. Arecombinant host cell, the host cell comprising the expression systemaccording to claim
 7. 9. A method of modulating expression ofsphingosine kinase, in a subject, said method comprising contacting thesphingosine kinase encoding gene with an effective amount of an agentfor a time and under conditions sufficient to modulate expression ofsphingosine kinase, said agent comprising an isolated polynucleotideaccording to claim 1.